Novel peptides and combination of peptides for use in immunotherapy against esophageal cancer and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/202,388, filed Jul. 5, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/188,870, filed Jul. 6, 2015, andGreat Britain Application No. 1511792.2, filed Jul. 6, 2015, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2016/065812 filed 5 Jul. 2016,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-051002_Sequence_Listing_ST25” created onApr. 27, 2018, and 16,290 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Esophageal cancer is the eighth most common cancer worldwide, with afive-year prevalence of 464,063 patients in 2012. Mortality rates arevery similar to incidence rates (400,169 versus 455,784 in 2012),pointing out the high fatality of esophageal cancer (World CancerReport, 2014; Ferlay et al., 2013; Bray et al., 2013).

Squamous cell carcinoma and adenocarcinoma represent the two most commonsubtypes of esophageal cancer. Both subtypes are more common in men thanin women, but they display distinct geographical distributions. Squamouscell carcinoma is more prevalent in low-resource regions withparticularly high incidence rates in the Islamic Republic of Iran, partsof China and Zimbabwe. Adenocarcinoma is the most common type ofesophageal cancer among Caucasians and populations with a highsocioeconomic status, with the United Kingdom, Australia, theNetherlands and the USA leading the way. The strongest risk factors forthe development of esophageal squamous cell carcinoma include alcoholand tobacco consumption, whereas esophageal adenocarcinoma is mainlyassociated with obesity and gastro-esophageal reflux disease. Incidencerates of esophageal adenocarcinoma are steadily rising in high-incomecountries, which might be attributed to increasing rates of obesity andgastro-esophageal reflux disease as well as to changes in theclassification of tumors at the gastro-esophageal junction.Neuroendocrine carcinoma, adenoid cystic carcinoma, adenosquamouscarcinoma, muco-epidermoid carcinoma, mixed adenoneuroendocrinecarcinoma, different sarcomas and melanoma represent rarer subtypes ofesophageal cancer (World Cancer Report, 2014).

The primary treatment strategy for esophageal cancer depends on tumorstage and location, histological type and the medical condition of thepatient. Surgery alone is not sufficient, except in a small subgroup ofpatients with squamous cell carcinoma. In general, surgery should becombined with pre- and eventually post-operative chemotherapy orpre-operative chemoradiation, while pre- or post-operative radiationalone was shown to confer no survival benefit. Chemotherapeutic regimensinclude oxaliplatin plus fluorouracil, carboplatin plus paclitaxel,cisplatin plus fluorouracil, FOLFOX and cisplatin plus irinotecan.Patients with HER2-positive tumors should be treated according to theguidelines for gastric cancer using a combination of cisplatin,fluorouracil and trastuzumab, as randomized data for targeted therapiesin esophageal cancer are very limited (Stahl et al., 2013; LeitlinieMagenkarzinom, 2012).

In general, most types of esophageal cancer are well manageable, ifpatients present with early-stage tumors, whereas therapeutic success isvery limited in later stages. Thus, development of new screeningprotocols could be very effective in reducing esophageal cancer-relatedmortality rates (World Cancer Report, 2014).

Immunotherapy might be a promising novel approach to treat advancedesophageal cancer. Several cancer-associated genes and cancer-testisantigens were shown to be over-expressed in esophageal cancer, includingdifferent MAGE genes, NY-ESO-1 and EpCAM (Kimura et al., 2007; Liang etal., 2005b; Inoue et al., 1995; Bujas et al., 2011; Tanaka et al., 1997;Quillien et al., 1997). Those genes represent very interesting targetsfor immunotherapy and most of them are under investigation for thetreatment of other malignancies (ClinicalTrials.gov, 2015). Furthermore,up-regulation of PD-L1 and PD-L2 was described in esophageal cancer,which correlated with poorer prognosis. Thus, esophageal cancer patientswith PD-L1-positive tumors might benefit from anti-PD-L1 immunotherapy(Ohigashi et al., 2005).

Clinical data on immunotherapeutic approaches in esophageal cancer arestill relatively scarce at present, as only a very limited number ofearly phase clinical trials have been completed (Toomey et al., 2013). Avaccine consisting of three peptides derived from three differentcancer-testis antigens (TTK protein kinase, lymphocyte antigen 6 complexlocus K and insulin-like growth factor (IGF)-II mRNA binding protein 3)was administered to patients with advanced esophageal cancer in a phaseI trial with moderate results (Kono et al., 2009). Intra-tumoralinjection of activated T cells after in vitro challenge with autologousmalignant cells and interleukin 2 elicited complete or partial tumorresponses in four of eleven patients in a phase I/I study (Toh et al.,2000; Toh et al., 2002). Further clinical trials are currently performedto evaluate the impact of different immunotherapies on esophagealcancer, including adoptive cellular therapy (NCT01691625, NCT01691664,NCT01795976, NCT02096614, NCT02457650) vaccination strategies(NCT01143545, NCT01522820) and anti-PD-L1 therapy (NCT02340975)(ClinicalTrials.gov, 2015).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and esophageal cancer in particular.There is also a need to identify factors representing biomarkers forcancer in general and esophageal cancer in particular, leading to betterdiagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 93 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 93, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 93,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1  Peptides according to the present invention SEQ ID No SequenceGene ID(s) Official Gene Symbol(s) 1 STYGGGLSV 3861, 3868 KRT14, KRT16 2SLYNLGGSKRISI   3852 KRT5 3 TASAITPSV   3852 KRT5 4 ALFGTILEL   2769GNA15 5 NLMASQPQL   5317 PKP1 6 LLSGDLIFL   2709 GJB5 7 SIFEGLLSGV  2709 GJB5 8 ALLDGGSEAYWRV  84985 FAM83A 9 HLIAEIHTA   5744 PTHLH 10SLDENSDQQV   6273 5100A2 11 ALWLPTDSATV   3914 LAMB3 12 GLASRILDA   3914LAMB3 13 SLSPVILGV  26525 IL36RN 14 RLPNAGTQV   3655 ITGA6 15 LLANGVYAA 55107 ANO1 16 VLAEGGEGV  10630 PDPN 17 MISRTPEV 2155, 28396, 3500, F7, IGHV4-31, IGHG1, 3501, 3502, 3503, IGHG2, IGHG3, IGHG4,   3507 IGHM18 FLLDQVQLGL  83882 TSPAN10 19 GLAPFLLNAV 101060689, 154761, FAM115C285966 20 IIEVDPDTKEML 100505503, 402057, RPS17L, RPS17P16, 442216, 6218RPS17P5, RPS17 21 IVREFLTAL  27297 CRCP 22 KLNDTYVNV  23306 TMEM194A 23KLSDSATYL No associated gene 24 LLFAGTMTV  29785 CYP2S1 25 LLPPPPPPA  9509 ADAMTS2 26 MLAEKLLQA   2195 FAT1 27 NLREGDQLL 113146 AHNAK2 28SLDGFTIQV   4939 0A52 29 SLDGTELQL 284114 TMEM102 30 SLNGNQVTV  79832OSER1 31 VLPKLYVKL 100996747, 441502, RPS26P11, RPS26, 6231, 643003,RPS26P28, RPS26P15, 644928, 728937, RPS26P25, RPS26P58 729188 32YMLDIFHEV   3038 HAS3 33 GLDVTSLRPFDL   2316 FLNA 34 SLVSEQLEPA  11187PKP3 35 LLRFSQDNA  51056 LAP3 36 FLLRFSQDNA  51056 LAP3 37 YTQPFSHYGQAL  6051 RNPEP 38 IAAIRGFLV  83451 ABHD11 39 LVRDTQSGSL    871 SERPINH1 40GLAFSLYQA    871 SERPINH1 41 GLESEELEPEEL   8106 PABPN1 42 TQTAVITRI 81610 FAM83D 43 KVVGKDYLL    832 CAPZB 44 ATGNDRKEAAENSL   7531 YWHAE45 MLTELEKAL   6279 S100A8 46 YTAQIGADIAL 64499, 7177 TPSB2, TPSAB1 47VLASGFLTV  79183 TTPAL 48 SMHQMLDQTL   7168 TPM1 49 GLMKDIVGA   8942KYNU 50 GMNPHQTPAQL    471 ATIC 51 KLFGHLTSA  57157 PHTF2 52VAIGGVDGNVRL   9948 WDR1 53 VVVTGLTLV    396 ARHGDIA 54 YQDLLNVKM1674, 4741, 4744, DES, NEFM, NEFH, NEFL, 4747, 7431, 9118 VIM, INA 55GAIDLLHNV 115362 GBPS 56 ALVEVTEHV  54972 TMEM132A 57 GLAPNTPGKA   9055PRC1 58 LILESIPVV   5597 MAPK6 59 SLLDTLREV   9989 PPP4R1 60 VVMEELLKV 23191 CYFIP1 61 TQTTHELTI   5093 PCBP1 62 ALYEYQPLQI   4331 MNAT1 63LAYTLGVKQL 158078, 1915, 1917  EEF1A1P5, EEF1A1, EEF1A2 64 GLTDVIRDV 80028 FBXL18 65 YVVGGFLYQRL   4074 M6PR 66 LLDEKVQSV 57616 TSHZ3 67SMNGGVFAV  23657 SLC7A11 68 PAVLQSSGLYSL 28396, 3500, 3501,IGHV4-31, IGHG1, IGHG2, 3502, 3503, 3507 IGHG3, IGHG4, IGHM 69GLLVGSEKVTM 3861, 3868, 644945 KRT14, KRT16, KRT16P3 70 FVLDTSESV1291, 1292 COL6A1, COL6A2 71 ASDPILYRPVAV   5315 PKM 72 FLPPAQVTV  65083NOL6 73 KITEAIQYV   6095 RORA 74 ILASLATSV  10844 TUBGCP2 75 GLMDDVDFKA 10525 HYOU1 76 KVADYIPQL   2744 GLS

TABLE 2  Additional peptides according to the presentinvention with no prior known cancer association Official Gene SEQ ID No Sequence Gene ID(s) Symbol(s) 77 VLVPYEPPQV 8626 TP63 78KVANIIAEV 5910 RAP1GDS1 79 GQDVGRYQV 6748 SSR4 80 ALQEALENA 9631 NUP15581 AVLPHVDQV 23379 KIAA0947 82 HLLGHLEQA 63977 PRDM15 83 ALADGVVSQA27238 GPKOW 84 SLAESLDQA 22894 DI53 85 NIIELVHQV 6850 SYK 86 GLLTEIRAV9263 STK17A 87 FLDNGPKTI 1982 E1F4G2 88 GLWEQENHL 79768 KATNBL1 89SLADSLYNL 23271 CAMSAP2 90 SIYEYYHAL 3091 HIF1A 91 KLIDDVHRL 6734 SRPR92 SILRHVAEV 1965 EIF251 93 VLINTSVTL 23036 ZNF292

TABLE 3  Peptides useful for e.g. personalized cancer therapies SEQOfficial Gene ID No Sequence Gene ID(s) Symbol(s) 94 TLLQEQGTKTV286887, 3852, KRT6C, KRT5, 3853, 3854 KRT6A, KRT6B 95 LIQDRVAEV  3914LAMB3 96 GAAVRIGSVL  9150 CTDP1 97 ELDRTPPEV 23450 5F3B3 98 VLFPNLKTV  646 BNC1 99 RVAPEEHPVL 440915, 60, POTEKP, ACTB, 641455, 71,POTEM, ACTG1, 728378 POTEF 100 GLYPDAFAPV  1991 ELANE 101 AMTQLLAGV 3371 TNC

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, lung cancer, urinarybladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellularcancer, renal cell cancer, brain cancer, colorectal cancer, breastcancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile ductcancer, prostate cancer and leukemia.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 93. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 76 (see Table 1), and their uses in theimmunotherapy of esophageal cancer, lung cancer, urinary bladder cancer,ovarian cancer, melanoma, uterine cancer, hepatocellular cancer, renalcell cancer, brain cancer, colorectal cancer, breast cancer, gastriccancer, pancreatic cancer, gallbladder cancer, bile duct cancer,prostate cancer and leukemia, and preferably esophageal cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID No. 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16,17, 18, 19, 25, 26, 30, 32, 34, 37, 40, 51, 55, 57, 58, 59, 62, 81, and82, and their uses in the immunotherapy of esophageal cancer, lungcancer, urinary bladder cancer, ovarian cancer, melanoma, uterinecancer, hepatocellular cancer, renal cell cancer, brain cancer,colorectal cancer, breast cancer, gastric cancer, pancreatic cancer,gallbladder cancer, bile duct cancer, prostate cancer and leukemia, andpreferably esophageal cancer. Further particularly preferred is thepeptide according to SEQ ID NO:9.

As shown in the following Table 4A, many of the peptides according tothe present invention are also found on other tumor types and can, thus,also be used in the immunotherapy of other indications. Also refer toFIGS. 1A-1V and Example 1.

Table 4A: Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases. The table shows for selected peptides on which additionaltumor types they were found and either over-presented on more than 5% ofthe measured tumor samples, or presented on more than 5% of the measuredtumor samples with a ratio of geometric means tumor vs normal tissuesbeing larger than 3. Over-presentation is defined as higher presentationon the tumor sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was testedwere: adipose tissue, adrenal gland, artery, bone marrow, brain, centralnerve, colon, duodenum, esophagus, gallbladder, heart, kidney, liver,lung, lymph node, mononuclear white blood cells, pancreas, peripheralnerve, peritoneum, pituitary, pleura, rectum, salivary gland, skeletalmuscle, skin, small intestine, spleen, stomach, thymus, thyroid gland,trachea, ureter, urinary bladder, vein.

SEQ ID No. Sequence Other relevant organs/diseases 2 SLYNLGGSKRISI NSCLC3 TASAITPSV NSCLC, Urinary bladder cancer 4 ALFGTILEL NSCLC 7 SIFEGLLSGVUrinary bladder cancer 8 ALLDGGSEAYWRV NSCLC, OC 9 HLIAEIHTA NSCLC 11ALWLPTDSATV NSCLC, Melanoma 12 GLASRILDA Urinary bladder cancer 13SLSPVILGV Uterine Cancer 15 LLANGVYAA HCC 17 MISRTPEV NSCLC, RCC, HCC 22KLNDTYVNV SCLC, Brain Cancer, HCC 26 MLAEKLLQA CRC 29 SLDGTELQL BRCA 31VLPKLYVKL GC 32 YMLDIFHEV Urinary bladder cancer 33 GLDVTSLRPFDL GC 34SLVSEQLEPA CRC, Urinary bladder cancer 35 LLRFSQDNA GC, HCC 36FLLRFSQDNA GC 37 YTQPFSHYGQAL GC, PC 38 IAAIRGFLV GC 39 LVRDTQSGSL GC 40GLAFSLYQA NSCLC, PC, BRCA, Urinary bladder cancer 41 GLESEELEPEEL GC 42TQTAVITRI GC 45 MLTELEKAL GC 46 YTAQIGADIAL GC 47 VLASGFLTVUrinary bladder cancer 49 GLMKDIVGA HCC 50 GMNPHQTPAQL GC 51 KLFGHLTSAGallbladder Cancer, Bile Duct Cancer 52 VAIGGVDGNVRL GC 54 YQDLLNVKMRCC, GC 55 GAIDLLHNV GC 56 ALVEVTEHV BRCA 57 GLAPNTPGKANSCLC, SCLC, BRCA, Melanoma 58 LILESIPVV NSCLC, Melanoma 61 TQTTHELTISCLC, Leukemia 62 ALYEYQPLQI NSCLC, HCC, Urinary bladder cancer 63LAYTLGVKQL GC 66 LLDEKVQSV Brain Cancer, Melanoma 67 SMNGGVFAVBrain Cancer, HCC 68 PAVLQSSGLYSL GC, PC 69 GLLVGSEKVTM PC 70 FVLDTSESVGC, HCC, Melanoma, OC 71 ASDPILYRPVAV GC, PC 72 FLPPAQVTVNSCLC, GC, HCC, Leukemia, Melanoma 73 KITEAIQYV BRCA 74 ILASLATSVCRC, HCC, Urinary bladder cancer 75 GLMDDVDFKABRCA, Melanoma, Urinary bladder cancer 76 KVADYIPQL NSCLC, SCLC 77VLVPYEPPQV NSCLC, Urinary bladder cancer 78 KVANIIAEV PC, Leukemia, OC80 ALQEALENA NSCLC, SCLC, Brain Cancer, CRC, HCC, Leukemia, BRCA, OC 81AVLPHVDQV Brain Cancer 82 HLLGHLEQA NSCLC, HCC, Leukemia, BRCA 83ALADGVVSQA Brain Cancer, GC, Melanoma, Urinary bladder cancer 85NIIELVHQV Leukemia 86 GLLTEIRAV Brain Cancer, Urinary bladder cancer 87FLDNGPKTI Brain Cancer, PC, OC 88 GLWEQENHL NSCLC, BRCA 89 SLADSLYNLBrain Cancer, BRCA, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer 91 KLIDDVHRL PC, PrC 92 SILRHVAEVNSCLC, CRC, HCC, Gallbladder Cancer, Bile Duct Cancer 94 TLLQEQGTKTVNSCLC NSCLC = non-small cell lung cancer, SCLC = small cell lung cancer,RCC = kidney cancer, CRC = colon or rectum cancer, GC = stomach cancer,HCC = liver cancer, PC = pancreatic cancer, PrC = prostate cancer,leukemia, BrCa = breast cancer

Table 4B: Peptides according to the present invention and their specificuses in other proliferative diseases, especially in other cancerousdiseases (amendment of Table 4). The table shows, like Table 4A, forselected peptides on which additional tumor types they were foundshowing over-presentation (including specific presentation) on more than5% of the measured tumor samples, or presentation on more than 5% of themeasured tumor samples with a ratio of geometric means tumor vs normaltissues being larger than 3. Over-presentation is defined as higherpresentation on the tumor sample as compared to the normal sample withhighest presentation. Normal tissues against which over-presentation wastested were: adipose tissue, adrenal gland, artery, bone marrow, brain,central nerve, colon, duodenum, esophagus, eye, gallbladder, heart,kidney, liver, lung, lymph node, mononuclear white blood cells,pancreas, parathyroid gland, peripheral nerve, peritoneum, pituitary,pleura, rectum, salivary gland, skeletal muscle, skin, small intestine,spleen, stomach, thyroid gland, trachea, ureter, urinary bladder, vein.

SEQ ID No Sequence Additional Entities 1 STYGGGLSVNSCLC, Melanoma, HNSCC 2 SLYNLGGSKRISI Urinary bladder cancer, HNSCC 3TASAITPSV HNSCC 4 ALFGTILELUrinary bladder cancer, Gallbladder Cancer, Bile Duct 5 NLMASQPQLCancer, AML, HNSCC HNSCC 6 LLSGDLIFL HNSCC 7 SIFEGLLSGVNSCLC, Gallbladder Cancer, Bile Duct Cancer, HNSCC 8 ALLDGGSEAYWRVUrinary bladder cancer, HNSCC 10 SLDENSDQQVUrinary bladder cancer, HNSCC 11 ALWLPTDSATVGallbladder Cancer, Bile Duct Cancer 13 SLSPVILGVNSCLC, Melanoma, Urinary bladder cancer, HNSCC 14 RLPNAGTQV Melanoma 15LLANGVYAA Urinary bladder cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer 16 VLAEGGEGVBrain Cancer, Melanoma, Gallbladder Cancer, Bile Duct Cancer, HNSCC 17MISRTPEV Urinary bladder cancer 18 FLLDQVQLGL Melanoma, NHL, HNSCC 19GLAPFLLNAV Melanoma, NHL, HNSCC 20 IIEVDPDTKEML HNSCC 22 KLNDTYVNV BRCA23 KLSDSATYL Melanoma 25 LLPPPPPPAGallbladder Cancer, Bile Duct Cancer, NHL, HNSCC 28 SLDGFTIQVBRCA, Melanoma, AML 29 SLDGTELQL Uterine Cancer, NHL 30 SLNGNQVTVBRCA, Melanoma, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, HNSCC 32 YMLDIFHEVGallbladder Cancer, Bile Duct Cancer, HNSCC 34 SLVSEQLEPA HNSCC 40GLAFSLYQA CRC, Melanoma, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer, HNSCC 42 TQTAVITRI HNSCC 48 SMHQMLDQTL GC 51 KLFGHLTSAGallbladder Cancer, Bile Duct Cancer 53 VVVTGLTLVGC, Urinary bladder cancer 55 GAIDLLHNV SCLC, Melanoma, NHL 56 ALVEVTEHVRCC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 57 GLAPNTPGKAUrinary bladder cancer, Uterine Cancer, HNSCC 58 LILESIPVVSCLC, CLL, Urinary bladder cancer, Uterine Cancer, NHL, HNSCC 59SLLDTLREV HNSCC 62 ALYEYQPLQISCLC, BRCA, Melanoma, OC, Gallbladder Cancer, Bile Duct Cancer, NHL 66LLDEKVQSV Urinary bladder cancer, HNSCC 67 SMNGGVFAVNSCLC, Gallbladder Cancer, Bile Duct Cancer, HNSCC 68 PAVLQSSGLYSL NHL69 GLLVGSEKVTM HNSCC 72 FLPPAQVTVCLL, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer, HNSCC74 ILASLATSV BRCA, HNSCC 75 GLMDDVDFKAGC, HCC, Gallbladder Cancer, Bile Duct Cancer, NHL, HNSCC 76 KVADYIPQLRCC, BRCA, Melanoma, Gallbladder Cancer, Bile Duct Cancer, NHL 77VLVPYEPPQV NHL, HNSCC 78 KVANIIAEV Urinary bladder cancer, HNSCC 79GQDVGRYQV SCLC, PC, PrC, CLL, BRCA, OC, Urinary bladder cancer, AML, NHL80 ALQEALENA Melanoma, Uterine Cancer, Gallbladder Cancer, BileDuct Cancer, HNSCC 81 AVLPHVDQV Uterine Cancer, NHL 82 HLLGHLEQA RCC 83ALADGVVSQA Uterine Cancer 84 SLAESLDQAMelanoma, Uterine Cancer, AML, NHL, HNSCC 85 NIIELVHQV CLL 86 GLLTEIRAVMelanoma, Gallbladder Cancer, Bile Duct Cancer, AML, NHL, HNSCC 87FLDNGPKTI Urinary bladder cancer, HNSCC 88 GLWEQENHLCRC, Uterine Cancer, AML, HNSCC 89 SLADSLYNL Melanoma 90 SIYEYYHALNHL, HNSCC 92 SILRHVAEV BRCA, Melanoma, AML, NHL

NSCLC=non-small cell lung cancer, SCLC=small cell lung cancer,RCC=kidney cancer, CRC=colon or rectum cancer, GC=stomach cancer,HCC=liver cancer, PC=pancreatic cancer, PrC=prostate cancer, BRCA=breastcancer, OC=ovarian cancer, NHL=non-Hodgkin lymphoma, AML=acute myeloidleukemia, CLL=chronic lymphocytic leukemia, HNSCC=head and neck squamouscell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 2, 3, 4, 7, 8, 9, 11, 13, 17, 40, 57, 58, 62, 67,72, 76, 77, 80, 82, 88, 92 and 94 for the—in one preferred embodimentcombined—treatment of non-small cell lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 18, 19, 25, 29, 55, 58, 62, 68, 75, 76, 77, 79, 81,84, 86, 90, and 92 for the—in one preferred embodimentcombined—treatment of lymphoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 22, 55, 58, 62, 57, 61, 76, 79 and 80 for the—in onepreferred embodiment combined—treatment of small-cell lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 17, 56, 76, 82, and 54 for the—in one preferredembodiment combined—treatment of renal cell cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 22, 66, 67, 80, 81, 83, 86, 87 and 89 for the—inone preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 31, 33, 35, 36, 37, 38, 39, 41, 42, 45, 46, 48, 50,52, 53, 54, 55, 63, 68, 70, 75 and 71 for the—in one preferredembodiment combined—treatment of gastric cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 26, 34, 40, 74, 80, 88, and 92 for the—in onepreferred embodiment combined—treatment of colorectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 15, 17, 22, 35, 49, 62, 67, 70, 72, 74, 75, 80, 82 and92 for the—in one preferred embodiment combined—treatment ofhepatocellular cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 37, 40, 68, 69, 71, 78, 79, 87 and 91 for the—in onepreferred embodiment combined—treatment of pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 79, and 91 for the—in one preferred embodimentcombined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 28, 58, 61, 72, 78, 79, 80, 82, 84, 86, 88, 92, and85 for the—in one preferred embodiment combined—treatment of leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 22, 28, 29, 30, 40, 56, 57, 62, 73, 74, 75, 76, 79,80, 82, 88, 92 and 89 for the—in one preferred embodimentcombined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 13, 14, 11, 16, 18, 19, 23, 28, 30, 40, 55, 57, 58,62, 66, 70, 72, 75, 76, 80, 84, 86, 89, 92, and 83 for the—in onepreferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 62, 70, 78, 79, 80 and 87 for the—in one preferredembodiment combined—treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 3, 4, 7, 8, 10, 12, 13, 15, 17, 30, 32, 34, 40, 47,53, 57, 58, 62, 66, 72, 74, 75, 77, 78, 79, 83, 86, 87, and 89 forthe—in one preferred embodiment combined—treatment of urinary bladdercancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 13, 15, 29, 30, 40, 56, 57, 58, 80, 81, 83, 84, and 88for the—in one preferred embodiment combined—treatment of uterinecancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 7, 11, 15, 16, 25, 30, 32, 40, 51, 56, 62, 67, 72,75, 76, 80, 86, 89 and 92 for the—in one preferred embodimentcombined—treatment of gallbladder and bile duct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. SEQ ID No 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, 16, 18, 19,20, 25, 30, 32, 34, 40, 42, 57, 58, 59, 66, 67, 69, 72, 74, 75, 77, 78,80, 84, 86, 87, 88, and 90 for the—in one preferred embodimentcombined—treatment of HNSCC.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofesophageal cancer, lung cancer, urinary bladder cancer, ovarian cancer,melanoma, uterine cancer, hepatocellular cancer, renal cell cancer,brain cancer, colorectal cancer, breast cancer, gastric cancer,pancreatic cancer, gallbladder cancer, bile duct cancer, prostate cancerand leukemia.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 93.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 93, preferably containing SEQ IDNo. 1 to SEQ ID No. 76, or a variant amino acid sequence. The presentinvention further relates to activated T cells, produced by the methodaccording to the present invention, wherein said T cell selectivelyrecognizes a cell which expresses a polypeptide comprising an amino acidsequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are esophageal cancer, lung cancer,urinary bladder cancer, ovarian cancer, melanoma, uterine cancer,hepatocellular cancer, renal cell cancer, brain cancer, colorectalcancer, breast cancer, gastric cancer, pancreatic cancer, gallbladdercancer, bile duct cancer, prostate cancer and leukemia, and preferablyesophageal cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably esophagealcancer. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally, the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

ABHD11 antisense RNA 1 (ABHD11-AS1) was described as a long noncodingRNA which was shown to be up-regulated in gastric cancer and associatedwith differentiation and Lauren histological classification. Thus,ABHD11-AS1 might be a potential biomarker for diagnosis of gastriccancer (Lin et al., 2014). ABHD11 activity was shown to be associatedwith the development of distant metastases in lung adenocarcinoma andthus might be a potential novel biomarker (Wiedl et al., 2011).

ADAMTS2 was shown to be dys-regulated in a patient with T/myeloid mixedphenotype acute leukemia (Tota et al., 2014). ADAMTS2 was described asbeing associated with the JNK pathway upon up-regulation through IL-6 inosteosarcoma cells (Alper and Kockar, 2014). ADAMTS2 may be a potentialdiagnostic marker for follicular thyroid cancer (Fontaine et al., 2009).ADAMTS2 was described as a potential marker of metastases in tonguesquamous cell carcinoma (Carinci et al., 2005). ADAMTS2 was shown to beup-regulated in renal cell carcinoma and to be associated with shorterpatient survival (Roemer et al., 2004). ADAMTS2 was shown to beregulated by the cell proliferation associated transforming growthfactor-beta 1 (Wang et al., 2003).

AHNAK2 encodes the scaffold protein AHNAK nucleoprotein 2 (Marg et al.,2010). AHNAK2 is an important element of the non-classical secretionpathway of fibroblast growth factor 1 (FGF1), a factor involved in tumorgrowth and invasion (Kirov et al., 2015). ANO1 encodes anoctamin 1, acalcium-activated chloride channel associated with small intestinalsarcoma and oral cancer (RefSeq, 2002). ANO1 is amplified in esophagealsquamous cell cancer (ESCC), gastrointestinal stromal tumor (GIST), headand neck squamous cell carcinoma (HNSCC), pancreatic and breast cancers(Qu et al., 2014).

ARHGDIA was shown to be down-regulated in hepatocellular carcinoma andupon breast cancer development (Liang et al., 2014; Bozza et al., 2015).ARHGDIA was shown to be associated with tumor invasion, metastasis,overall survival and time to recurrence in hepatocellular carcinoma.Thus, ARHGDIA may provide a potential therapeutic target forhepatocellular carcinoma (Liang et al., 2014). ARHGDIA was shown to bedown-regulated in the lung cancer cell line A549 upon periplocintreatment. Periplocin-inhibited growth of the lung cancer cells thus maybe associated with ARHGDIA (Lu et al., 2014). ARHGDIA knock-down wasshown to be associated with increased apoptosis in lung-derived normaland cultured tumor cells. Thus, ARHGDIA was described as a negativeregulator of apoptosis which may represent a potential therapeutictarget (Gordon et al., 2011). ARHGDIA was described to be associatedwith ovarian clear cell and high-grade serous carcinoma staging (Canetet al., 2011). ARHGDIA was described as an apoptotic pathway relatedgene which was shown to be de-regulated in fibrosarcoma HT1080 cellsupon TRAIL-mediated apoptosis (Daigeler et al., 2008). ARHGDIA was shownto be up-regulated in the oxaliplatin-resistent colonic cancer cell lineTHC8307/L-OHP and was described as a gene involved in anti-apoptosis.Thus, ARHGDIA may be a potential marker associated with oxaliplatinsensitivity (Tang et al., 2007). ARHGDIA over-expression was shown to beregulated by the putative tumor suppressor ACVR2, a member of thecancer-related TGFBR2 family, in a wild-type ACVR2 transfected MSI-Hcolon cancer cell line carrying an ACVR2 frameshift mutation (Deacu etal., 2004). ARHGDIA was described as a key regulator of the Rho GTPases.ARHGDIA depletion was shown to induce constitutive activation of RhoGTPases and COX-2 pathways in association with breast cancer progressionin a breast cancer xenograft animal model (Bozza et al., 2015). ARHGDIAsignaling was shown to be de-regulated in colorectal cancer (Sethi etal., 2015). ARHGDIA was shown to target MEK1/2-Erk upon SUMOylation,which is associated with inhibition of C-Jun/AP-1, cyclin dltranscription, and cell cycle progression. Thus, ARHGDIA is associatedwith suppression of cancer cell growth (Cao et al., 2014). ARHGDIA wasdescribed as a novel suppressor in prostate cancer which may play acritical role in regulating androgen receptor signaling and prostatecancer growth and progression (Zhu et al., 2013b).

ATIC was described as potential gene fusion partner of thecancer-associated anaplastic lymphoma kinase in anaplastic larger celllymphoma (Cheuk and Chan, 2001). ATIC was shown to be presented as achimeric fusion with ALK in an inflammatory myofibroblastic tumor of theurinary bladder (Debiec-Rychter et al., 2003). Inhibition of theaminoimidazole carboxyamide ribonucleotide transformylase (AICAR)activity of ATIC in a model breast cancer cell line was shown to resultin dose-dependent reduction of cell numbers and cell division rates.Thus, ATIC may be a potential target in cancer therapy (Spurr et al.,2012).

CAPZB was reported to be over-expressed in human papillomaviruses18-positive oral squamous cell carcinomas and was identified as prostatecancer susceptibility locus (Lo et al., 2007; Nwosu et al., 2001).

COL6A1 is up-regulated in the reactive stroma of castration-resistantprostate cancer and promotes tumor growth (Zhu et al., 2015). Col6A1 isover-expressed in CD166-pancreatic cancer cells that show strongerinvasive and migratory activities than those of CD166+ cancer cells(Fujiwara et al., 2014). COL6A1 is highly expressed in bone metastasis(Blanco et al., 2012). COL6A1 was found to be up-regulated in cervicaland ovarian cancer (Zhao et al., 2011; Parker et al., 2009). COL6A1 isdifferentially expressed in astrocytomas and glioblastomas (Fujita etal., 2008).

COL6A2 is associated with cervical cancer, poor overall survival inhigh-grade serous ovarian cancer, B-precursor acute lymphoblasticleukemia, hepatocellular carcinoma, primary and metastatic brain tumors,squamous cell carcinoma of the lung, head and neck squamous cellcarcinoma and was described as a potential DNA methylation for cervicalcancer (Cheon et al., 2014; Chen et al., 2014d; Vachani et al., 2007;Liu et al., 2010; Seong et al., 2012; Hogan et al., 2011).

CYFIP1 was shown to be down-regulated during invasion of epithelialtumors (Silva et al., 2009). CYFIP1 down-regulation is associated withpoor prognosis in epithelial tumors (Silva et al., 2009).

CYP2S1 was shown to regulate colorectal cancer growth in the cell lineHCT116 through association with PGE2-mediated activation of β-cateninsignaling (Yang et al., 2015b). CYP2S1 was described as beingup-regulated in multiple epithelial-derived cancers and in hypoxic tumorcells (Nishida et al., 2010; Madanayake et al., 2013). CYP2S1 depletionin bronchial epithelial cell lines was shown to result in alteredregulation in key pathways implicated in cell proliferation andmigration such as the mTOR signal pathway (Madanayake et al., 2013).CYP2S1 depletion was shown to be associated with drug sensitivity incolorectal and breast cancer (Tan et al., 2011). CYP2S1 was shown to becorrelated with survival in breast cancer and is associated with poorprognosis in colorectal cancer (Murray et al., 2010; Kumarakulasinghamet al., 2005). CYP2S1 was shown to metabolize BaP-7,8-diol into thehighly mutagenic and carcinogenicbenzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide and thus may play animportant role in benzo[a]pyrene-induced carcinogenesis (Bui et al.,2009). CYP2S1 was shown to be significantly up-regulated in ovariancancer metastasis compared with primary ovarian cancer (Downie et al.,2005).

DES expression in colorectal cancer stroma was shown to be correlatedwith advanced stage disease (Arentz et al., 2011). DES was shown to beup-regulated in colorectal cancer (Ma et al., 2009). DES was shown to beassociated with severity and differentiation of colorectal cancer and adecreased survival rate (Ma et al., 2009). DES was described as apotential oncofetal serum tumor marker for colorectal cancer (Ma et al.,2009). DES was shown to be a specific marker for rhabdomyosarcoma(Altmannsberger et al., 1985). DES was described as one of three membersof a protein panel that may be potentially useful for staging of bladdercarcinoma by using immunohistochemistry (Council and Hameed, 2009).

DIS3 was shown to be frequently mutated in multiple myeloma andrecurrently mutated in acute myeloid leukemia (Ding et al., 2012; Lohret al., 2014). DIS3 mutations in multiple myeloma were shown to beassociated with shorter median overall survival. Mutations in minorsubclones were shown to be associated with worse response to therapycompared to patients with DIS3 mutations in the major subclone(Weissbach et al., 2015). DIS3 was shown to be up-regulated incolorectal carcinomas through a 13q gain. Silencing of DIS3 was shown toaffect important tumorigenic characteristics such as viability,migration and invasion. Thus, DIS3 may be a novel candidate oncogenecontributing to colorectal cancer progression (de Groen et al., 2014).DIS3 was described as part of a gene panel which may be used incombination with plasma protein based biomarkers to allow earlierdiagnosis of epithelial ovarian cancer (Pils et al., 2013). DIS3 may bea potential candidate gene for breast cancer susceptibility sincenumerous polymorphisms were detected upon mutation screening in breastcancer families (Rozenblum et al., 2002).

EEF1A1 was shown to be up-regulated in a variety of cancer entities,including colorectal cancer, ovarian cancer, gastric cancer, prostatecancer, glioblastoma and squamous cell carcinoma and was described aspotential serum biomarker for prostate cancer (Lim et al., 2011; Qi etal., 2005; Matassa et al., 2013; Vui-Kee et al., 2012; Kuramitsu et al.,2010; Kido et al., 2010; Scrideli et al., 2008; Rehman et al., 2012).Mechanistically, EEF1A1 inhibits apoptosis through an interaction withp53 and p73, promotes proliferation by transcriptional repression of thecell cycle inhibitor p21 and participates in the regulation ofepithelial-mesenchymal transition (Blanch et al., 2013; Choi et al.,2009; Hussey et al., 2011).

EEF1A2 was described as being up-regulated in breast cancer, ovariancancer, lung cancer, pancreatic cancer, gastric cancer, prostate cancerand TFE3 translocation renal cell carcinoma (Pflueger et al., 2013; Sunet al., 2014; Yang et al., 2015c; Zang et al., 2015; Abbas et al.,2015). EEF1A2 was shown to be associated with poor prognosis in ovariancancer, gastric cancer, pancreatic ductal adenocarcinoma and lungadenocarcinoma (Duanmin et al., 2013; Yang et al., 2015c; Li et al.,2006; Lee and Surh, 2009). EEF1A2 was described as being associated withoncogenesis since it stimulates the phospholipid signaling and activatesthe Akt-dependent cell migration and actin remodeling, which ultimatelyfavors tumorigenesis (Abbas et al., 2015). EEF1A2 was described toinhibit p53 function in hepatocellular carcinoma viaPI3K/AKT/mTOR-dependent stabilization of MDM4. Strong activation of theEEF1A2/PI3K/AKT/mTOR/MDM4 signaling pathway was shown to be associatedwith short survival in hepatocellular carcinoma and thus may be a targetfor therapy in a subset of patients (Longerich, 2014). EEF1A2 was shownto be associated with TNM stage, invasiveness and survival in pancreaticcancer patients. Thus, EEF1A2 might be a potential target for treatmentof pancreatic cancer (Zang et al., 2015). EEF1 A2 was shown to beassociated with prostate cancer development through promotion ofproliferation and inhibition of apoptosis and thus might serve as apotential therapeutic target in prostate cancer (Sun et al., 2014).EEF1A2 was shown to interact with the tumor suppressor protein p16,which leads to down-regulation of EEF1A2 and is associated withinhibition of cancer cell growth (Lee et al., 2013). EEF1A2 was show tobe associated with nodal metastasis and perineural invasion inpancreatic ductal adenocarcinoma (Duanmin et al., 2013). EEF1A2 wasshown to be associated with survival in breast cancer (Kulkarni et al.,2007). EEF1A2 was described as a putative oncogene and tumor suppressorgene in lung adenocarcinoma cell lines and in ovarian cancer (Lee, 2003;Zhu et al., 2007a).

EIF2S1 was described as a promoter of tumor progression and resistanceto therapy upon phosphorylation. However, EIF2S1 was also described tobe implicated in suppressive effects on tumorigenesis (Zheng et al.,2014). EIF2S1 was described as a downstream effector of mTOR whichdecreases survival of cancer cells upon excessive phosphorylation andthus may serve as a target for drug development (Tuval-Kochen et al.,2013).

EIF4G2 was described as one gene of a set of core genes that were shownto be associated with the elimination of tumor formation of pediatricglioma CD133+ cells (Baxter et al., 2014). EIF4G2 was shown to beassociated with repression of diffuse large B cell lymphoma developmentupon down-regulation through miR-520c-3p (Mazan-Mamczarz et al., 2014).EIF4G2 was shown to facilitate protein synthesis and cell proliferationby modulating the synthesis of cell cycle proteins (Lee and McCormick,2006). EIF4G2 was shown to be down-regulated in bladder tumors anddown-regulation was associated with invasive tumors (Buim et al., 2005).EIF4G2 was described as being involved in MycN/IFNgamma-inducedapoptosis and both viability and death of neuroblastoma cells (Wittke etal., 2001).

F7 in complex with tissue factor was described as being aberrantlyexpressed on the surface of cancer cells, including ovarian cancer. Thecomplex was further described as being associated with the induction ofmalignant phenotypes in ovarian cancer (Koizume and Miyagi, 2015). TheF7-tissue factor complex pathway was described as a mediator of breastcancer progression which may stimulate the expression of numerousmalignant phenotypes in breast cancer cells. Thus, the F7-tissue factorpathway is a potentially attractive target for breast cancer treatment(Koizume and Miyagi, 2014). F7 was shown to be regulated by the androgenreceptor in breast cancer (Naderi, 2015). F7 was shown to be associatedwith the regulation of autophagy via mTOR signaling in hepatocellularcarcinoma cell lines (Chen et al., 2014a). F7 was shown to be associatedwith tumor invasion and metastasis in colorectal cancer and ovariancancer (Tang et al., 2010; Koizume et al., 2006). F7 was shown to beectopically up-regulated in colorectal cancer (Tang et al., 2009). F7 incomplex with the tissue factor was shown to be associated withchemotherapy resistance in neuoblastoma (Fang et al., 2008a).

FAM115C is up-regulated upon hypoxia in non-small cell lung cancer(Leithner et al., 2014).

FAM83A was described as a potential biomarker for lung cancer (Li etal., 2005). FAM83A was described as a marker gene which can be used in apanel with NPY1R and KRT19 to detect circulating cancer cells in breastcancer patients (Liu et al., 2014d). FAM83A ablation from breast cancercells was shown to result in diminished MAPK signaling with markedsuppression of growth in vitro and tumorigenicity in vivo (Cipriano etal., 2014). Furthermore, the FAM83 protein family was described as anovel family of oncogenes which regulates MAPK signaling in cancer andthus is suitable for the development of cancer therapies that aim atsuppressing MAPK signaling (Cipriano et al., 2014). FAM83A was shown tobe associated with trastuzumab resistance in HER2-positive breast cancercell lines (Boyer et al., 2013). In general, FAM83A was shown to be acandidate gene associated with EGFR-tyrosine kinase inhibitor resistancein breast cancer (Lee et al., 2012). FAM83A was described as beingassociated with poor prognosis in breast cancer (Lee et al., 2012).FAM83A was shown to be up-regulated in non-small cell lung cancer (Qu etal., 2010). FAM83A was shown to be a potential specific and sensitivemarker to detect circulating tumor cell in the peripheral blood ofnon-small cell lung cancer patients (Qu et al., 2010).

Up-regulation of FAM83D affects the proliferation and invasion ofhepatocellular carcinoma cells (Wang et al., 2015a; Liao et al., 2015b).FAM83D is significantly elevated in breast cancer cell lines and inprimary human breast cancers (Wang et al., 2013b).

FAT1 was described as being significantly mutated in squamous-cellcancer of the head and neck, frequently mutated in cervicaladenocarcinoma, bladder cancer, early T-cell precursor acutelymphoblastic leukemia, fludarabine refractoriness chronic lymphocyticleukemia, glioblastoma and colorectal cancer and mutated in esophagealsquamous cell carcinoma (Gao et al., 2014; Neumann et al., 2013; Morriset al., 2013; Messina et al., 2014; Mountzios et al., 2014; Cazier etal., 2014; Chung et al., 2015). FAT1 was described as being repressed inoral cancer and preferentially down-regulated in invasive breast cancer(Katoh, 2012). FAT1 was described as being up-regulated in leukemiawhich is associated with a poor prognosis in preB-acute lymphoblasticleukemia (Katoh, 2012). FAT1 was shown to be up-regulated in pancreaticadenocarcinoma and hepatocellular carcinoma (Valletta et al., 2014;Wojtalewicz et al., 2014). FAT1 was described to suppress tumor growthvia activation of Hippo signaling and to promote tumor migration viainduction of actin polymerization (Katoh, 2012). FAT1 was shown to be acandidate cancer driver gene in cutaneous squamous cell carcinoma(Pickering et al., 2014). FAT1 was described as a tumor suppressor whichis associated with Wnt signaling and tumorigenesis (Morris et al.,2013).

Depending on its subcellular localization, filamin A plays a dual rolein cancer: In the cytoplasm, filamin A functions in various growthsignaling pathways, in addition to being involved in cell migration andadhesion pathways. Thus, its over-expression has a tumor-promotingeffect. In contrast to full-length filamin A, the C-terminal fragment,which is released upon proteolysis of the protein, localizes to thenucleus, where it interacts with transcription factors and therebysuppresses tumor growth and metastasis (Savoy and Ghosh, 2013).

A tumor-specific C-terminal truncation of GBP5 was described as beingpotentially responsible for the dys-regulation of GBP5 in lymphoma cells(Wehner and Herrmann, 2010). GBP5 was described to possess possiblecancer-related functions because of the restricted expression pattern ofthe three GBP5 splice variants in cutaneous T-cell lymphoma tumortissues and cell lines as well as in melanoma cell lines (Fellenberg etal., 2004).

GJB5 was shown to be down-regulated in non-small cell lung cancer celllines, larynx cancer and head and neck squamous cell carcinomas (Zhanget al., 2012; Broghammer et al., 2004; Al Moustafa et al., 2002). GJB5was described to act as a tumor suppressor in non-small cell lung cancercell lines through inhibition of cell proliferation and metastasis(Zhang et al., 2012). GJB5 was shown to be up-regulated in sessileserrated adenomas/polyps, premalignant lesions that may account for20-30% of colon cancers (Delker et al., 2014). GJB5 expression wasdescribed as significantly altered during skin tumor promotion andprogression in a mouse model (Slaga et al., 1996).

GLS was described as being indirectly regulated by the MYC oncogene inorder to increase glutamine metabolism in cancer cells (Dang et al.,2009). GLS was shown to be suppressed by the tumor suppressor NDRG2 incolorectal cancer (Xu et al., 2015). GLS was described as beingup-regulated in pancreatic ductal adenocarcinomas, triple-negativebreast cancer, hepatocellular carcinoma, oral squamous cell carcinoma,colorectal cancer and malignant glia-derived tumors (van Geldermalsen etal., 2015; Szeliga et al., 2014; Huang et al., 2014a; Cetindis et al.,2015; Yu et al., 2015a; Chakrabarti et al., 2015). GLS was shown to beassociated with survival in hepatocellular carcinoma and was alsodescribed as a sensitive and specific biomarker for pathologicaldiagnosis and prognosis of hepatocellular carcinoma (Yu et al., 2015a).Loss of one copy of GLS was shown to blunt tumor progression in animmune-competent MYC-mediated mouse model of hepatocellular carcinoma(Xiang et al., 2015). GLS was shown to be required for tumorigenesis andinhibition of tumor-specific GLS was described as a potential approachfor cancer therapy (Xiang et al., 2015). GLS was shown to be associatedwith Taxol-resistance in breast cancer (Fu et al., 2015a). GLSover-expression was shown to be highly correlated with tumor stage andprogression in prostate cancer patients (Pan et al., 2015). GLSexpression was shown to be associated with deeper tumor infiltration andpathological patterns of tubular adenocarcinoma in colon cancertumorigenesis. GLS may serve as a target for colorectal cancer therapy(Huang et al., 2014a). Silencing of the GLS isoenzyme KGA was shown toresult in lower survival ratios in the glioma cell lines SFxL and LN229(Martin-Rufian et al., 2014). Silencing of GLS in the glioma cell linesSFxL and LN229 was also shown to result in induction of apoptosis byevoking lower c-myc and bcl-2 expression, as well as higherpro-apoptotic bid expression (Martin-Rufian et al., 2014). ErbB2activation was shown to up-regulate GLS expression via the NF-kBpathway, which promoted breast cancer cell proliferation (Qie et al.,2014). Knock-down or inhibition of GLS in breast cancer cells with highGLS levels was shown to result in significantly decreased proliferation(Qie et al., 2014).

GNA15 was shown to be up-regulated in primary and metastatic smallintestinal neuroendocrine neoplasias (Zanini et al., 2015). Increasedexpression of GNA15 was shown to be associated with a poorer survival,suggesting that GNA15 may have a pathobiological role in smallintestinal neuroendocrine neoplasias and thus could be a potentialtherapeutic target (Zanini et al., 2015). GNA15 was described as beingdown-regulated in many non-small cell lung cancer cell lines (Avasaralaet al., 2013). High expression of GNA15 in normal karyotype acutemyeloid leukemia was shown to be associated with a significant pooreroverall survival (de Jonge et al., 2011). GNA15 was shown to be acritical downstream effector of non-canonical Wnt signaling and aregulator of non-small cell lung cancer cell proliferation andanchorage-independent cell growth. Thus, GNA15 is a potentialtherapeutic target for the treatment of non-small cell lung cancer(Avasarala et al., 2013). GNA15 was shown to be associated withtumorigenic signaling in pancreatic carcinoma (Giovinazzo et al., 2013).GNA15 was shown to stimulate STAT3 via a c-Src/JAK- and ERK-dependentmechanism upon constitutive activation in human embryonic kidney 293cells (Lo et al., 2003).

HAS3 under-expression was shown to be associated with advanced tumorstage, nodal metastasis, vascular invasion and poorer disease-specificsurvival and metastasis-free survival in urothelial carcinoma of theupper urinary tract and the urinary bladder (Chang et al., 2015). Thus,HAS3 may serve as a prospective prognostic biomarker and a noveltherapeutic target in urothelial carcinomas (Chang et al., 2015). HAS3was shown to favor pancreatic cancer growth by hyaluronan accumulation(Kultti et al., 2014). HAS3 inhibition was shown to decrease viabilityin the colorectal adenocarcinoma cell line SW620 (Heffler et al., 2013).HAS3 inhibition was shown to be associated with differential expressionof several genes involved in the regulation of SW620 colorectal tumorcell survival (Heffler et al., 2013). HAS3 was shown to be associatedwith the mediation of colon cancer growth by inhibiting apoptosis (Tenget al., 2011). HAS3 was shown to be up-regulated in esophageal squamouscell carcinoma, adenocarcinomas and squamous cell carcinomas of the lungand nodular basal cell carcinoma (Tzellos et al., 2011; Twarock et al.,2011; de Sa et al., 2013). HAS3 was described as an independentprognostic factor in breast cancer since HAS3 expression in stromalcells of breast cancer patients was shown to be correlated with a highrelapse rate and short overall survival (Auvinen et al., 2014). HAS3 wasshown to be associated with serous ovarian carcinoma, renal clear cellcarcinoma, endometrioid endometrial carcinoma and osteosarcoma (Nykoppet al., 2010; Weiss et al., 2012; Cai et al., 2011; Tofuku et al.,2006).

HIF1A was shown to be associated with tumor necrosis in aggressiveendometrial cancer. HIF1A was further described to be a potential targetfor the treatment of this disease (Bredholt et al., 2015). HIF1A wasshown to be associated with hepatocarcinogenesis, sarcoma metastasis andnasopharyngeal carcinoma (Chen et al., 2014c; El-Naggar et al., 2015; Liet al., 2015b). A single nucleotide polymorphism in HIF1A was shown tobe significantly associated with clinical outcomes of aggressivehepatocellular carcinoma patients after surgery (Guo et al., 2015).Aberrant HIF1A activity together with aberrant STAT3 activity was shownto drive tumor progression in malignant peripheral nerve sheath tumorcell lines. Thus, inhibition of the STAT3/HIF1A/VEGF-A signaling axiswas described as a viable treatment strategy (Rad et al., 2015). HIF1Awas described as an important target for hypoxia-driven drug resistancein multiple myeloma (Maiso et al., 2015). HIF1A was shown to beasymmetrical expressed in three different cell lines that correspondwith the stages of multiple myeloma pathogenesis, suggesting that HIF1Ais involved in the tumorigenesis and metastasis of multiple myeloma(Zhao et al., 2014b). The long noncoding HIF1A antisense RNA-2 wasdescribed as being up-regulated in non-papillary clear-cell renalcarcinomas and gastric cancer and is associated with tumor cellproliferation and poor prognosis in gastric cancer (Chen et al., 2015b).De-regulation of the PI3K/AKT/mTOR pathway through HIF1A was describedto be critical for quiescence, maintenance and survival of prostatecancer stem cells (Marhold et al., 2015). HIF1A was described as onegene of a 4-gene classifier which is prognostic for stage I lungadenocarcinoma (Okayama et al., 2014). A polymorphism of HIF1A was shownto be associated with increased susceptibility to digestive tract cancerin Asian populations (Xu et al., 2014). HIF1A was described as aprognostic marker in sporadic male breast cancer (Deb et al., 2014).

The activity of intracellular HYOU1 protein has been shown to provide asurvival benefit in cancer cells during tumor progression or metastasis.The extracellular HYOU1 protein plays an essential role in thegeneration of an anti-tumor immune response by facilitating the deliveryof tumor antigens for their cross-presentation (Fu and Lee, 2006; Wanget al., 2014). HYOU1 protein has been introduced in cancer immunotherapyand showed a positive immunomodulating effect (Yu et al., 2013; Chen etal., 2013; Yuan et al., 2012; Wang and Subjeck, 2013).

A study has shown that IGHG1 was over-expressed in human pancreaticcancer tissues compared to adjacent non-cancerous tissues. On thecontrary, the IGHG1 protein was down-regulated in infiltrating ductalcarcinomas tissues (Kabbage et al., 2008; Li et al., 2011). siRNAtargeted silencing of IGHG1 was able to inhibit cell viability andpromote apoptosis (Pan et al., 2013).

Researchers have observed expression of IGHG3 in Saudi females affectedby breast cancer. Similarly, gains in copy number as well as elevatedlevels of IGHG3 were detected in African American men suffering fromprostate cancer. Another report showed that IGHG3 expression is found insquamous non-small cell lung cancers, malignant mesothelioma as well ason tumor cells that are sporadically seen in MALT lymphomas and thatshow a propensity for differentiation into plasma cells (Remmelink etal., 2005; Bin Amer et al., 2008; Ledet et al., 2013; Zhang et al.,2013; Sugimoto et al., 2014).

IGHG4 encodes immunoglobulin heavy constant gamma 4 (G4m marker) and islocated on chromosome 14q32.33 (RefSeq, 2002). Recent work has detectedrearrangements involving IGHG4 in primary testicular diffuse large Bcell lymphoma (Twa et al., 2015). IGHM encodes immunoglobulin heavyconstant mu (RefSeq, 2002). Studies have observed down-regulation ofIGHM in Chinese patients affected by rhabdomyosarcoma. Others havedetected expression of IGHM in diffuse large B-cell lymphoma. Anothergroup has found that in diffuse large B-cell lymphoma the IGHM gene isconserved only on the productive IGH allele in most IgM+ tumors. Inaddition, epithelioid angiomyolipoma samples did not show any reactivityfor transcription factor binding to IGHM enhancer 3 or transcriptionfactor EB (Kato et al., 2009; Blenk et al., 2007; Ruminy et al., 2011;Liu et al., 2014b).

IL36RN was described as a marker which can significantly distinguishstage III from stages I and II in lung adenocarcinoma (Liang et al.,2015).

Reduced expression of INA was shown to be associated with metastases,recurrence and shorter overall survival in pancreatic neuroendocrinetumors. Thus, INA may be a useful prognostic biomarker for pancreaticneuroendocrine tumor aggressiveness (Liu et al., 2014a). INA wasdescribed as being up-regulated in oligodendroglia phenotype gliomas andINA expression was shown to be correlated with progression-free survivalof oligodendrogliomas and glioblastomas (Suh et al., 2013). INA wasdescribed as a marker for neuroblastoma which is useful for thedifferential diagnostic work-up of small round cell tumors of childhood(Willoughby et al., 2008).

ITGA6 expression is up-regulated in different cancer entities includingbreast, prostate, colon and gastric cancer and is associated with tumorprogression and cell invasion (Mimori et al., 1997; Lo et al., 2012;Haraguchi et al., 2013; Rabinovitz et al., 1995; Rabinovitz andMercurio, 1996). The proliferative effects of the Abeta4 variant ofITGA6 appear to be mediated through the Wnt/beta-catenin pathway (Groulxet al., 2014). The Abeta4 variant of ITGA6 leads to the VEGF-dependentactivation of the PI3K/Akt/mTOR pathway. This pathway plays an importantrole in the survival of metastatic carcinoma cells (Chung et al., 2002).

KRT14 was highly expressed in various squamous cell carcinomas such asesophageal, lung, larynx, uterine cervical as well as in adenomatoidodontogenic tumor. However, it was absent in small cell carcinoma of theurinary bladder and weak in lung adenocarcinoma, gastric adenocarcinoma,colorectal adenocarcinoma, hepatocellular carcinoma, pancreatic ductaladenocarcinoma, breast infiltrating dutal adenocarcinoma, thyroidpapillary carcinoma and uterine endometrioid adenocarcinoma (Xue et al.,2010; Terada, 2012; Vasca et al., 2014; Hammam et al., 2014; Shruthi etal., 2014). In bladder cancer, KRT14 expression was strongly associatedwith poor survival (Volkmer et al., 2012).

Over-expression of KRT16 was found in basal-like breast cancer celllines as well as in carcinoma in situ. Others did not find significantdifference in immunohistochemical expression of KRT16 betweennon-recurrent ameloblastomas and recurrent ameloblastomas (Joosse etal., 2012; Ida-Yonemochi et al., 2012; Safadi et al., 2016). Inaddition, in silico analyses showed correlation between KRT16 expressionand shorter relapse-free survival in metastatic breast cancer (Joosse etal., 2012).

KRT5 was shown to be up-regulated in breast cancers of young women(Johnson et al., 2015). KRT5 was shown to be associated with inferiordisease-free survival in breast cancer in young women and withunfavorable clinical outcome in premenopausal patients with hormonereceptor-positive breast cancer (Johnson et al., 2015; Sato et al.,2014). KRT5 was shown to be regulated by the tumor suppressor BRCA1 inthe breast cancer cell lines HCC1937 and T47D (Gorski et al., 2010).KRT5 was shown to be de-regulated in malignant pleural mesothelioma(Melaiu et al., 2015). KRT5 was described as a diagnostic mesothelialmarker for malignant mesothelioma (Arif and Husain, 2015). KRT5 wasshown to be correlated with the progression of endometrial cancer (Zhaoet al., 2013). KRT5 was shown to be mutated and down-regulated ininvasive tumor areas in a patient with verrucous carcinoma (Schumann etal., 2012). KRT5 was shown to be part of a four-protein panel which wasdifferentially expressed in colorectal cancer biopsies compared tonormal tissue samples (Yang et al., 2012). KRT5 and three other proteinsof the four-protein panel were described as novel markers and potentialtargets for treatment for colorectal cancer (Yang et al., 2012). KRT5was described as being associated with basal cell carcinoma (Depianto etal., 2010). KRT5 was described as a candidate to identify urothelialcarcinoma stem cells (Hatina and Schulz, 2012).

Activation of the kynurenine pathway, in which KYNU is involved, wasshown to be significantly higher in glioblastoma and suggests theinvolvement of the kynurenine pathway in glioma pathophysiology (Adamset al., 2014). KYNU was described as cancer-linked gene whose expressionwas altered upon aryl hydrocarbon receptor knock-down in the MDA-MB-231breast cancer cell line (Goode et al., 2014). KNYU was shown to bedifferentially expressed in high and non-aggressive osteosarcoma celllines, suggesting that it might have an important role in the process ofosteosarcoma tumorigenesis. Thus, KYNU might also represent a candidatefor a future therapeutic targets (Lauvrak et al., 2013). KYNU was shownto be associated with the re-expression of tumorigenicity innon-tumorigenic HeLa and human skin fibroblast hybrid cells. Thus, KYNUmay provide an interesting candidate for the regulation of tumorigenicexpression (Tsujimoto et al., 1999).

Transcription analysis of LAMB3 in combination with two other genes wasshown to be useful in the diagnosis of papillary thyroid carcinoma andprediction of lymph node metastasis risk (Barros-Filho et al., 2015).LAMB3 was shown to be associated with the cancer entities of oralsquamous cell carcinoma, prostate cancer, gastric cancer, colorectalcancer, ewing family tumors, lung carcinoma, breast carcinoma andovarian carcinoma (Volpi et al., 2011; Ii et al., 2011; Reis et al.,2013; Stull et al., 2005; Irifune et al., 2005; Tanis et al., 2014).LAMB3 was shown to be up-regulated in cervical squamous cell carcinoma,lung cancer, gastric cancer, nasopharyngeal carcinoma and esophagealsquamous cell carcinoma (Kwon et al., 2011; Wang et al., 2013a; Yamamotoet al., 2013; Kita et al., 2009; Fang et al., 2008b). LAMB3 wasdescribed as a protein known to influence cell differentiation,migration, adhesion, proliferation and survival and which functions asan oncogene in cervical squamous cell carcinoma (Yamamoto et al., 2013).Knock-down of LAMB3 was shown to suppress lung cancer cell invasion andmetastasis in vitro and in vivo. Thus, LAMB3 is a key gene which playsan important role in the occurrence and metastasis of lung cancer (Wanget al., 2013a). LAMB3 was shown to be regulated by the tumor suppressormiR-218 in head and neck squamous cell carcinoma (Kinoshita et al.,2012). Silencing of LAMB3 in head and neck squamous cell carcinoma wasshown to result in inhibition of cell migration and invasion (Kinoshitaet al., 2012). LAMB3 expression was shown to be correlated with thedepth of invasion and venous invasion in esophageal squamous cellcarcinoma (Kita et al., 2009). Methylation of LAMB3 was shown to becorrelated with several parameters of poor prognosis in bladder cancer(Sathyanarayana et al., 2004).

Inhibition of LAP3 was shown to result in suppressed invasion in theovarian cancer cell line ES-2 through down-regulation of fascin andMMP-2/9. Thus, LAP3 may act as a potential anti-metastasis therapeutictarget (Wang et al., 2015d). High expression of LAP3 was shown to becorrelated with grade of malignancy and poor prognosis of gliomapatients (He et al., 2015). LAP3 was shown to promote glioma progressionby regulating cell growth, migration and invasion and thus might be anew prognostic factor (He et al., 2015). Frameshift mutations in genesinvolved in amino acid metabolism including LAP3 were detected inmicrosatellite instability-high gastric and colorectal cancer (Oh etal., 2014). LAP3 was shown to be up-regulated in hepatocellularcarcinoma, esophageal squamous cell carcinoma and prostate cancer (Zhanget al., 2014; Tian et al., 2014; Lexander et al., 2005). LAP3 was shownto promote hepatocellular carcinoma cells proliferation by regulatingG1/S checkpoint in cell cycle and advanced cells migration (Tian et al.,2014). Expression of LAP3 was further shown to be correlated withprognosis and malignant development of hepatocellular carcinoma (Tian etal., 2014). Silencing of LAP3 in the esophageal squamous cell carcinomacell line ECA109 was shown to reduce cell proliferation and colonyformation while LAP3 knock-down resulted in cell cycle arrest (Zhang etal., 2014). Over-expression of LAP3 in the esophageal squamous cellcarcinoma cell line TE1 was shown to favor cell proliferation andinvasiveness (Zhang et al., 2014). Thus, LAP3 was shown to play a rolein the malignant development of esophageal squamous cell carcinoma(Zhang et al., 2014).

Researchers have reported expression of M6PR in colon carcinoma celllines as well as in choriocarcinoma cells (Braulke et al., 1992;O'Gorman et al., 2002). In breast cancer, low-level expression of M6PRwas associated with poor patient prognosis (Esseghir et al., 2006).Furthermore, over-expression of M6PR resulted in a decreased cellulargrowth rate in vitro and decreased tumor growth in nude mice (O'Gormanet al., 2002).

MAPK6 was shown to play a role in regulating cell morphology andmigration in the breast cancer cell line MDA-MB-231 (Al-Mahdi et al.,2015). MAPK6 was described as a part of the cancer-associated MAPKsignaling pathway, being associated with BRAF and MEK1/2 signaling inmelanoma (Lei et al., 2014; Hoeflich et al., 2006). MAPK6 was shown tobe up-regulated in lung carcinoma, gastric cancer and oral cancer (Longet al., 2012; Rai et al., 2004; Liang et al., 2005a). MAPK6 was shown topromote lung cancer cell invasiveness by phosphorylating the oncogeneSRC-3. Thus, MAPK6 may be an attractive target for therapeutic treatmentof invasive lung cancer (Long et al., 2012). MAPK6 was described as apotential target for the anti-cancer drug development against drugresistant breast cancer cells (Yang et al., 2010). Over-expression ofMAPK6 in gastric carcinomas was shown to be correlated with TNM staging,serosa invasion and lymph node involvement (Liang et al., 2005a). MAPK6was shown to be a binding partner of the core cell cycle machinerycomponent cyclin D3, suggesting that MAPK6 has a potential activity incell proliferation (Sun et al., 2006).

MNAT1 was shown to be associated with poor prognosis in estrogenreceptor-positive/HER2-negative breast cancer (Santarpia et al., 2013).Loss of intrinsic fragmentation of MNAT1 during granulopoiesis was shownto promote the growth and metastasis of leukemic myeloblasts (Lou etal., 2013). MNAT1 was shown to be dys-regulated in the ovarian cancercell line OAW42 upon knock-down of the putative oncogene ADRM1 (Fejzo etal., 2013). MNAT1 gene silencing mediated by siRNA was shown to suppresscell growth of the pancreatic cancer cell line BxPC3 in vitro, andsignificantly achieved an anti-tumor effect on a subcutaneouslytransplanted pancreatic tumor in vivo (Liu et al., 2007a). Geneticvariants in MNAT1 were described to be associated with thesusceptibility of lung cancer (Li et al., 2007). Infection of thepancreatic cancer cell line BxPC3 with a recombinant adenovirus encodingantisense MNAT1 was shown to result in decreased expression of MNAT1 andan increased proportion of G0/G1 phase cells. Thus, MNAT1 is suggestedto play an important role in the regulation of cell cycle G1 to Stransition in the pancreatic cancer cell line BxPC3 (Zhang et al.,2005). MNAT1-modulated cyclin-dependent kinase-activating kinaseactivity was shown to cross-regulate neuroblastoma cell G1 arrest and iscrucial in the switch from proliferation to differentiation inneuroblastoma cells (Zhang et al., 2004).

DNA methylation-associated silencing of NEFH in breast cancer was shownto be frequent, cancer-specific, and correlated with clinical featuresof disease progression (Calmon et al., 2015). NEFH was further describedto be inactivated through DNA methylation in pancreatic, gastric andcolon cancer and thus might contribute to the progression of thesemalignancies as well (Calmon et al., 2015). NEFH CpG island methylationwas shown to be associated with advanced disease, distant metastasis andprognosis in renal cell carcinoma (Dubrowinskaja et al., 2014). Thus,NEFH methylation could be a candidate epigenetic marker for theprognosis of renal cell carcinoma (Dubrowinskaja et al., 2014). NEFH wasshown to be up-regulated in extraskeletal myxoid chondrosarcoma of thevulva (Dotlic et al., 2014). Over-expression of NEFH in a hepatocellularcarcinoma cell line was shown to reduce cell proliferation whileknock-down of NEFH promoted cell invasion and migration in vitro, andincreased the ability to form tumors in mice. Thus, NEFH functions as atumor suppressor in hepatocellular carcinoma (Revill et al., 2013). NEFHwas shown to be frequently methylated in Ewing sarcoma and thus might beassociated with tumorigenesis (Alholle et al., 2013).

DNA methylation-mediated silencing of NEFL was shown to be a frequentevent in breast cancer that may contribute to the progression of breastcancer and possibly other malignancies such as pancreatic, gastric andcolon cancer (Calmon et al., 2015). NEFL was described as a potentialtumor suppressor gene which is associated with the cancer of severalorgans (Huang et al., 2014c). NEFL was described to potentially play arole in cancer cell apoptosis and invasion in head and neck squamouscell carcinoma cell lines (Huang et al., 2014c). NEFL methylation wasdescribed as a novel mechanism that conferred cisplatin chemoresistancein head and neck cancer cell lines through interaction with the mTORsignaling pathway (Chen et al., 2012). NEFL was described as a candidatebiomarker predictive of chemotherapeutic response and survival inpatients with head and neck cancer (Chen et al., 2012). High expressionof NEFL was described to be correlated with better clinical outcome insupratentorial ependymoma (Hagel et al., 2013). NEFL was shown to beectopically expressed in breast cancer and decreased in primary breastcancers with lymph node metastases compared to cancers with negativelymph nodes (Li et al., 2012). Low expression of NEFL was shown toindicate poor five-year disease-free survival for early-stage breastcancer patients and thus could be a potential prognostic factor forearly-stage breast cancer patients (Li et al., 2012). NEFL was shown tobe down-regulated in glioblastoma multiforme (Khalil, 2007). Allelicdeletion at chromosome 8p21-23, where NEFL is located, was described asan early and frequent event in the carcinogenesis and development oflung cancer and was also described to be associated with breast cancer,prostate cancer and hepatitis B virus-positive hepatocellular carcinoma(Seitz et al., 2000; Becker et al., 1996; Haggman et al., 1997; Kurimotoet al., 2001).

NEFM was described as a gene with relevance to tumor progression andassociation with the processes involved in metastasis (Singh et al.,2015). NEFM was shown to be hypo-methylated and up-regulated inesophageal cancer (Singh et al., 2015). NEFM was described as acandidate tumor suppressor gene that is frequently down-regulated inglioblastoma (Lee et al., 2015a). DNA methylation-associated silencingof NEFM in breast cancer was shown to be frequent, cancer-specific, andcorrelated with clinical features of disease progression (Calmon et al.,2015). NEFM was further described to be inactivated through DNAmethylation in pancreatic, gastric and colon cancer and thus mightcontribute to the progression of these malignancies as well (Calmon etal., 2015). NEFM was shown to be associated with prostate cancer andastrocytomas (Wu et al., 2010; Penney et al., 2015). NEFM was describedas a novel candidate tumor suppressor gene which was shown to bemethylated in renal cell carcinoma (Ricketts et al., 2013). Methylationof NEFM was shown to be associated with prognosis in renal cellcarcinoma (Ricketts et al., 2013). NEFM was described as a potentialdiagnostic marker which was shown to be differentially expressed inneuroendocrine tumor cell lines compared to non-neuroendocrine tumorcell lines (Hofsli et al., 2008).

NUP155 was described as a potential epigenetic biomarker of white bloodcell's DNA which is associated with breast cancer predisposition(Khakpour et al., 2015). NUP155 was described as strictly required forthe proliferation and survival of NUP214-ABL1-positive T-cell acutelymphoblastic leukemia cells and thus constitutes a potential drugtarget in this disease (De et al., 2014).

OAS2 was shown to be associated with impairment of the CD3-zeta chainexpression through caspase-3 activation. Deficiency of the CD3-zetachain was described to be often observed in oral cancer (Dar et al.,2015). OAS2 was described to be involved in a sub-pathway of theadvanced prostate cancer risk-associated innate immunity andinflammation pathway (Kazma et al., 2012). Sub-pathway analysis revealedthat OAS2 is nominally associated with advanced prostate cancer risk(Kazma et al., 2012).

Lower expression of PABPN1 in non-small cell lung cancer was shown to becorrelated with a poor prognosis (Ichinose et al., 2014). Loss of PABPN1was described to potentially promote tumor aggressiveness by releasingcancer cells from microRNA-mediated gene regulation in non-small celllung cancer (Ichinose et al., 2014). A N-terminal polyalanine expansionmutant of PABPN1 was shown to be associated with induction of apoptosisvia the p53 pathway in the HeLa and HEK-293 cell lines (Bhattacharjee etal., 2012).

PCBP1 was described to be central to cancer stem cells enrichment andfunctionality in prostate cancer cells (Chen et al., 2015a). PCBP1 wasdescribed as an inhibitor of gastric cancer pathogenesis whosedown-regulation is associated with the malignant phenotype in bothcultured and xenograft gastric cancer cells (Zhang et al., 2015e). Basedon the differential expression between benign and malignant serum andtissue samples, respectively, in patients with serous adenocarcinoma ofthe ovary, PCBP1 was suggested to play a role in ovarian cancerpathophysiology (Wegdam et al., 2014). PCBP1 was shown to be animportant mediator of TGF-1-induced epithelial-mesenchymal transition, apre-requisite for tumor metastasis, in the gall bladder carcinoma cellline GBC-SD (Zhang and Dou, 2014). PCBP1 expression levels were shown toregulate the capacity of the gall bladder carcinoma cell line GBC-SD tomigrate and invade in vitro (Zhang and Dou, 2014). Thus, PCBP1 might bea potential prognostic marker for gall bladder carcinoma metastasis(Zhang and Dou, 2014). PCBP1 down-regulation was described as beingpotentially involved with cervical cancer pathogenesis (Pathak et al.,2014). PCBP1 was described as a regulator of the tumorsuppression-associated transcription factor p63 (Cho et al., 2013). Highexpression of PCBP1 in complete hydatidiform moles was shown to beassociated with lower risk of progression to gestational trophoblastictumors, while PCBP1 expression was significantly lower in malignanttransformed moles (Shi et al., 2012). Thus, PCBP1 was suggested to playan important role in the pathogenesis of gestational trophoblastictumors (Shi et al., 2012). Over-expression of PCBP1 was shown to resultin suppression of the translation of the metastasis associated PRL-3protein and inactivation of AKT whereas knock-down of PCBP1 was shown tocause activation of AKT and promotion of tumorigenesis (Wang et al.,2010). PCBP1 was described to play a negative role in tumor invasion inthe hepatoma cell line HepG2 (Zhang et al., 2010). Loss of PCBP1 inhuman hepatic tumor was described to contribute to the formation of ametastatic phenotype (Zhang et al., 2010).

PDPN was described to be up-regulated in squamous cell carcinomas,mesotheliomas, glioblastomas and osteosarcomas (Fujita and Takagi,2012). PDPN was described as a regulator of tumor invasion andmetastasis since PDPN is associated with several pathways whichparticipate in epithelial-to-mesenchymal transition, collective-cellmigration, platelet activation, aggregation, and lymphangiogenesis (Danget al., 2014). PDPN was described as a marker in oral carcinogenesis andepithelioid mesotheliomas (Swain et al., 2014; Ordonez, 2005). PDPNup-regulation was described to be associated with lymph node metastasisand poor prognosis in squamous cell carcinoma of the upper aerodigestivetract (Chuang et al., 2013). PDPN was described to be expressed invascular tumors, malignant mesothelioma, tumors of the central nervoussystem, germ cell tumors, squamous cell carcinomas and aggressive tumorswith higher invasive and metastatic potential (Raica et al., 2008).Thus, PDPN might be considered as an attractive therapeutic target fortumor cells (Raica et al., 2008).

PHTF2 was shown to be down-regulated in lingual squamous cell carcinoma(Huang et al., 2007).

PKM2 was shown to be crucial for cancer cell proliferation and tumorgrowth (Chen et al., 2014b; Li et al., 2014; DeLaBarre et al., 2014).N-myc acts as a transcriptional regulator for PKM2 in medulloblastoma(Tech et al., 2015). PKM2 seems to play a role in hepatocarcinogenesis,epithelial mesenchymal transition, and angiogenesis (Nakao et al.,2014). PKM2 is one of the two key factors of the Warburg effect inoncology (Tamada et al., 2012; Warner et al., 2014; Ng et al., 2015).Expression of PKM2 is up-regulated in cancer cells (Chaneton andGottlieb, 2012; Luo and Semenza, 2012; Wu and Le, 2013). In malignantcells PKM2 functions in glycolysis, as a transcriptional coactivator andas a protein kinase. In the latter function it translocates to thenucleus and phosphorylates histone 3 which finally causes the progressof the cell cycle in glioblastomas (Semenza, 2011; Luo and Semenza,2012; Tamada et al., 2012; Venneti and Thompson, 2013; Yang and Lu,2013; Gupta et al., 2014; Iqbal et al., 2014; Chen et al., 2014b; Warneret al., 2014). The low-activity-dimeric PKM2 might play a role in cancerinstead of the active tetrameric form (Mazurek, 2011; Wong et al., 2015;Iqbal et al., 2014; Mazurek, 2007).

PKP1 was shown to be down-regulated in prostate cancer and esophagealadenocarcinoma (Kaz et al., 2012; Yang et al., 2015a). Knock-down ofPKP1 in the non-neoplastic, prostatic BPH-1 cell line led to reducedapoptosis and differential expression of genes such as the prostatecancer-associated SPOCK1 gene (Yang et al., 2015a). Collectively,altered expression of PKP1 and SPOCK1 appears to be frequent andcritical event in prostate cancer and PKP1 is suggested to have atumor-suppressive function (Yang et al., 2015a). Reduced expression ofPKP1 was shown to be associated with significantly shorter time to onsetof distant metastasis in oral cavity squamous cell carcinoma (Harris etal., 2015). PKP1 loss through promoter methylation was described to beassociated with the progression of Barrett's esophagus to esophagealadenocarcinoma (Kaz et al., 2012). PKP1 was shown to be up-regulated innon-small cell lung cancer and may be a good marker to distinguishsquamous-cell carcinomas samples (Sanchez-Palencia et al., 2011). PKP1was shown to be up-regulated in the well-differentiated liposarcoma cellline GOT3 (Persson et al., 2008). Decreased PKP1 expression wasdescribed to promote increased motility in head and neck squamous cellcarcinoma cells (Sobolik-Delmaire et al., 2007). PKP1 loss was shown tobe associated with cervical carcinogenesis (Schmitt-Graeff et al.,2007). PKP1 was shown to be associated with local recurrences ormetastases as well as poor survival in patients with squamous cellcarcinoma of the oropharynx (Papagerakis et al., 2003).

Increased PKP3 mRNA in the blood of gastrointestinal cancer patients canbe used as a biomarker and predictor for disease outcome(Valladares-Ayerbes et al., 2010). Over-expression of PKP3 wascorrelated with a poor outcome in breast, lung and prostate cancer,whereas down-regulation in bladder cancer is linked to invasive behavior(Furukawa et al., 2005; Breuninger et al., 2010; Demirag et al., 2012;Takahashi et al., 2012). Loss of PKP3 leads to increased protein levelsof MMP7 and PRL3, which are required for cell migration and tumorformation (Khapare et al., 2012; Basu et al., 2015).

Knock-down of PPP4R1 was shown to suppress cell proliferation in thebreast cancer cell line ZR-75-30 (Qi et al., 2015). Thus, PPP4R1 couldpromote breast cancer cell proliferation and might play a vital role inbreast cancer occurrence (Qi et al., 2015). Knock-down of PPP4R1 in thehepatocellular carcinoma cell line HepG2 was shown to result indecreased cell proliferation, colony formation and cell cycle arrest atG2/M (Wu et al., 2015). Knock-down of PPP4R1 was further shown to leadto the inactivation of the p38 and c-Jun N-terminal kinase signalingcascades in HepG2 cells, which indicates that PPP4R1 could promote cellproliferation (Wu et al., 2015). Thus, PPP4R1 plays a crucial role inpromoting hepatocellular carcinoma cell growth (Wu et al., 2015). PPP4R1was described as a negative regulator of inhibitor of NF-kB kinaseactivity in lymphocytes whose down-regulation promotes oncogenic NF-kBsignaling in a subgroup of T cell lymphomas (Brechmann et al., 2012).

PRC1 was described to be associated with radioresistance in cervicalcancer since cervical cancer tissues showed a high differentialexpression of PRC1 after radiation (Fu et al., 2015b). A genetic loci inintron 14 of PRC1 was described to be associated with breast cancersusceptibility (Cai et al., 2014). PRC1 was described as one gene of afive-gene signature which could be proposed as a prognostic signaturefor disease free survival of breast cancer patients (Mustacchi et al.,2013). PRC1 was shown to be up-regulated in ovarian carcinoma, cervicalcancer and bladder cancer (Espinosa et al., 2013; Ehrlichova et al.,2013; Kanehira et al., 2007). PRC1 was shown to be up-regulated duringthe 4-hydroxy-estradiol-mediated malignant transformation of the mammaryepithelial cell line MCF-10A (Okoh et al., 2013). PRC1 was described asa gene with significant biological implications in tumor pathogenesiswhich can be used in a gene-set to predict the prognosis of resectablepatients with non-small cell lung cancer upon adjuvant chemotherapy(Tang et al., 2013). PRC1 was suggested to be negatively regulated bythe cell cycle associated kinase Plk1 (Hu et al., 2012). Knock-down ofPRC1 in the bladder cancer cell line NIH3T3 was shown to result in asignificant increase of multinuclear cells and subsequent cell death(Kanehira et al., 2007). Furthermore, PRC1 was shown to interact withthe novel cancer-testis antigen MPHOSPH1 in bladder cancer cells and theMPHOSPH1/PRC1 complex was suggested to play a crucial role in bladdercarcinogenesis and could be a novel therapeutic target (Kanehira et al.,2007). PRC1 was shown to be regulated by p53 (Li et al., 2004).

Expressed sequence tag profiling identified PRDM15 as an up-regulatedgene in lymphomas (Giallourakis et al., 2013). PRDM15 was described as acandidate tumor suppressor gene which may contribute to the developmentor progression of pancreatic cancer (Bashyam et al., 2005).

Distinct polymorphisms in PTHLH were shown to be associated with lungcancer risk and prognosis (Manenti et al., 2000). Up-regulation of PTHLHin a C57BL/6-mouse-derived model of spontaneously metastatic mammarycancer was described as potentially being involved in metastaticdissemination of breast cancer (Johnstone et al., 2015). PTHLH was shownto be up-regulated in oral squamous cell carcinoma, chondroid neoplasms,adult T-cell leukemia/lymphoma and clear cell renal cell carcinomas(Bellon et al., 2013; Yang et al., 2013a; Yao et al., 2014; Lv et al.,2014). PTHLH up-regulation was shown to be associated with poorpathologic differentiation and poor prognosis in patients with head andneck squamous cell carcinoma (Lv et al., 2014). PTHLH was shown to beup-regulated through p38 MAPK signaling, which contributes to coloncancer cell extravasation of the lung by caspase-independent death inendothelial cells of the lung microsvasculature (Urosevic et al., 2014).PTHLH was shown to be significantly differentially expressed in squamouscell carcinoma compared with normal skin (Prasad et al., 2014). PTHLHwas described as a part of a four-gene signature associated withsurvival among patients with early-stage non-small cell lung cancer(Chang et al., 2012). Disruption of anti-proliferative function byframeshift mutations of PTHLH was described to contribute to thedevelopment of early colorectal cancer in patients with hereditarynon-polyposis colorectal cancer (Yamaguchi et al., 2006). PTHLHup-regulation was shown to be associated with poor outcome both inoverall survival and disease-free survival for clear cell renal cellcarcinoma patients who underwent nephrectomy (Yao et al., 2014). PTHLHwas shown to positively modulate cell cycle progression and to changethe expression of proteins involved in cell cycle regulation via ERK1/2,p38, MAPK, and PI3K signaling pathways in the colorectal adenocarcinomacell line Caco-2 (Calvo et al., 2014).

RAP1GDS1 was shown to promote proliferation of pancreatic cancer cells(Schuld et al., 2014). Simultaneous loss of two splice variants ofRAP1GDS1 in non-small cell lung carcinoma cell line NCI-H1703 xenograftsin mice was shown to result in decreased tumorigenesis (Schuld et al.,2014). RAP1GDS1 was shown to promote cell cycle progression in multipletypes of cancer, making it a valuable target for cancer therapeutics(Schuld et al., 2014). RAP1GDS1 was shown to be up-regulated in breastcancer, prostate cancer and non-small cell lung carcinoma (Hauser etal., 2014; Tew et al., 2008; Zhi et al., 2009). The SmgGDS-558 splicevariant of RAP1GDS1 was shown to be a unique promoter of RhoA and NF-kBactivity which plays a functional role in breast cancer malignancy(Hauser et al., 2014). High RAP1GDS1 expression was shown to beassociated with worse clinical outcome in breast cancer (Hauser et al.,2014). RAP1GDS1 was shown to regulate cell proliferation, migration, andNF-kappaB transcriptional activity in non-small cell lung cancer,therefore promoting the malignant phenotype of this disease. Thus,RAP1GDS1 is an intriguing therapeutic target in non-small cell lungcancer (Tew et al., 2008). RAP1GDS1 was shown to be fused to NUP98 inT-cell acute lymphoblastic leukemia (Romana et al., 2006).

RNPEP activity was shown to be up-regulated in colorectal adenomas,papillary thyroid carcinoma, breast cancer and clear cell renal cellcarcinoma (Ramirez-Exposito et al., 2012; Larrinaga et al., 2013; Perezet al., 2015; Varona et al., 2007). RNPEP was shown to be associatedwith the tumor growth of rat C6 gliomas implanted at the subcutaneousregion (Mayas et al., 2012).

RORA was described as a potential lung cancer oncogene (Wang et al.,2015e). RORA was shown to be associated with the expression of thepotential tumor suppressor gene OPCML in colon cancer (Li et al.,2015a). Two single nucleotide polymorphisms in RORA were shown to beassociated with breast cancer (Truong et al., 2014). RORA was describedas a potential tumor suppressor and therapeutic target for breast cancer(Du and Xu, 2012). RORA was shown to be down-regulated in colorectaladenocarcinomas and breast cancer (Kottorou et al., 2012; Du and Xu,2012). Stable over-expression of RORA in the hepatoma cell line HepG2was shown to influence the expression of genes involved in glucosemetabolism and liver carcinogenesis, indicating an association of RORAwith the carcinogenesis within cells of hepatic origin (Chauvet et al.,2011). RORA was shown to be differentially methylated in gastric cancercompared to normal gastric mucosa (Watanabe et al., 2009). RORA wasdescribed to be associated with the control of cell growth anddifferentiation, and with the control of metastatic behavior in theandrogen-independent prostate cancer cell line DU 145 (Moretti et al.,2002).

RPS17 was shown to be differentially expressed in metastatic uvealmelanoma in normal whole blood and tissues prone to metastaticinvolvement by uveal melanoma, suggesting that RPS17 might play a rolein tropism of uveal melanoma metastasis (Demirci et al., 2013). RPS17was shown to be up-regulated in hepatocellular carcinoma (Liu et al.,2007b).

Knock-down of RPS26 was shown to induce p53 stabilization andactivation, resulting in p53-dependent cell growth inhibition (Cui etal., 2014). RPS26 was further shown to play a role in DNA damageresponse by directly influencing p53 transcriptional activity (Cui etal., 2014).

S100A2 was shown to be associated with non-small cell lung cancer andwas described as a predictive marker for poor overall survival in lungsquamous carcinoma patients (Hountis et al., 2014; Zhang et al., 2015d).S100A2 was described as a down-stream target of the oncogene KRAS and apromoter of tumor progression in lung cancer (Woo et al., 2015). S100A2was described as a promising marker for the prediction of overallsurvival in pancreatic ductal adenocarcinoma (Jamieson et al., 2011).Altered expression of S100A2 through nitrosamine N-nitrosopyrrolidonewas described as a potential reason for the tumor progression ofsquamous cell carcinomas of the esophagus among black South Africans(Pillay et al., 2015). S100A2 was shown to be up-regulated in the earlystage of non-small cell lung cancer, in plasma of nasopharyngealcarcinoma patients, laryngeal cancer, gastric cancer and epidermaltumors (Zhu et al., 2013a; Lin et al., 2013; Zhang et al., 2015a; Zha etal., 2015; Wang et al., 2015c). Methylation-associated inactivation ofS100A2 was shown to be frequent in head and neck and bladder cancer, andthus may be an important event in the tumorigenesis of these diseases(Lee et al., 2015c). S100A2 cytoplasmic expression was shown to beup-regulated in oral squamous cell carcinoma, while the nuclearexpression was down-regulated (Kumar et al., 2015). Cytoplasmicup-regulation of S100A2 was shown to be a potential predictor ofrecurrence risk in oral squamous cell carcinoma patients (Kumar et al.,2015). S100A2 was described to play a role in the metastasis of mammarycarcinomas (Naba et al., 2014). S100A2 was shown to be a BRCA1/p63co-regulated tumor suppressor gene which plays a role in the regulationof mutant p53 stability by modulating the binding of mutant p53 to HSP90(Buckley et al., 2014). S100A2 was described as a candidate tumorsuppressor gene which is down-regulated in recurrent nasopharyngealcancer, and thus may play an important role in the occurrence ofrecurrent nasopharyngeal cancer (Huang et al., 2014b). S100A2 was shownto be down-regulated in gastric cancer and down-regulation was shown tobe associated with advanced depth of invasion, lymph node metastasis,decreased relapse-free probability, and decreased overall survival (Liuet al., 2014e). Thus, S100A2 down-regulation may be a negativeindependent prognostic biomarker for gastric cancer (Liu et al., 2014e).S100A2 was further shown to negatively regulate the MEK/ERK signalingpathway in MGC-803 cancer cells (Liu et al., 2014e). Over-expression ofS100A2 was shown to induce epithelial-mesenchymal transition in A549lung cancer cells followed by increased invasion and enhanced Aktphosphorylation and increased tumor growth in immunocompromised mice(Naz et al., 2014). Protumorigenic actions of S100A2 were furtherdescribed to involve regulation of PI3/Akt signaling and functionalinteraction with the TGF beta signaling way protein Smad3 (Naz et al.,2014). Expression of S100A2 was shown to be correlated with histologicalgrade, lymph node metastasis, clinical stage, and a poor survival rateof patients with perihilar and extra-hepatic cholangiocarcinoma (Sato etal., 2013). Thus, S100A2 may function as a prognostic marker incholangiocarcinoma patients (Sato et al., 2013).

S100A8 was described as an important mediator in acute and chronicinflammation which interacts with myeloid-derived suppressor cells in apositive feedback loop to promote tumor development and metastasis(Zheng et al., 2015). S100A8 was described as a potential diagnosticbiomarker, prognostic indicator and therapeutic target in non-small celllung cancer (Lim and Thomas, 2013). Over-expression of S100A8 was shownto be associated with stage progression, invasion, metastasis and poorsurvival in bladder cancer (Yao et al., 2007). S100A8 was shown to be adiagnostic marker for invasive bladder carcinoma (Ismail et al., 2015).S100A8 was shown to be up-regulated in anaplastic thyroid carcinoma,giant cell tumor of bone and colorectal cancer (Reeb et al., 2015; Zhanget al., 2015b; Liao et al., 2015a). In vivo analysis in mice usinganaplastic thyroid carcinoma cells with S100A8 knock-down revealedreduced tumor growth and lung metastasis, as well as significantlyprolonged animal survival (Reeb et al., 2015). S100A8 was shown topromote anaplastic thyroid carcinoma cell proliferation throughinteraction with RAGE, which activates the p38, ERK1/2 and JNK signalingpathways in tumor cells (Reeb et al., 2015). Thus, S100A8 couldrepresent a relevant therapeutic target in anaplastic thyroid carcinoma(Reeb et al., 2015). S100A8 was shown to be associated with high-riskchronic lymphocytic leukemia (Alsagaby et al., 2014). S100A8 was shownto be associated with kidney cancer progression and was described as aprospective biomarker and therapeutic target for kidney cancer (Mirza etal., 2014). S100A8 was described as a part of calprotectin, which is aheterodimer that is required for the progression of non-inflammationdriven liver tumor and might represent a therapeutic target for thetreatment of hepatocellular carcinoma (De et al., 2015). S100A8 wasshown to regulate colon cancer cell cycle and proliferation by inductionof Id3 expression while inhibiting p21 (Zhang et al., 2015b).

SERPINH1 encodes serpin peptidase inhibitor, clade H (heat shock protein47), member 1, (collagen binding protein 1), a serine proteinaseinhibitor. SERPINH1 functions as a collagen-specific molecular chaperonein the endoplasmic reticulum (RefSeq, 2002). SERPINH1 is over-expressedin many human cancers, including stomach cancer, lung cancer, pancreaticductal adenocarcinoma, glioma, and ulcerative colitis-associatedcarcinomas (Zhao et al., 2014a). SERPINH1 was shown to be up-regulatedin hepatocellular carcinoma, esophageal squamous cell carcinoma,cholangiocellular carcinoma, stomach cancer, lung cancer, pancreaticductal adenocarcinoma, ulcerative colitis-associated carcinomas andglioma (Zhao et al., 2014a; Padden et al., 2014; Lee et al., 2015b;Naboulsi et al., 2015). Over-expression of SERPINH1 was shown to beassociated with poor prognosis in patients with esophageal squamous cellcarcinoma and the level of immunostaining of SERPINH1 and pathologicstage were shown to be significantly correlated with overall andrecurrence-free survival (Lee et al., 2015b). Thus, SERPINH1 may be apotential prognostic biomarker for esophageal squamous cell carcinoma(Lee et al., 2015b). Knock-down of SERPINH1 in glioma cells was shown toinhibit glioma cell growth, migration and invasion in vitro whileSERPINH1 knock-down in vivo was shown to inhibit tumor growth andinduced apoptosis (Zhao et al., 2014a). Thus, SERPINH1 could be atherapeutic target for the treatment of glioma (Zhao et al., 2014a).SERPINH1 was shown to be down-regulated in metastases compared to theprimary tumor of oral squamous cell carcinomas with multiple lymph nodeinvolvement, indicating that SERPINH1 might be associated with themetastatic potential of these tumors (Nikitakis et al., 2003).

SLC7A11 was shown to be down-regulated in drug resistant variants of theW1 ovarian cancer cell line and thus might play a role in cancer celldrug resistance (Januchowski et al., 2013). SLC7A11 was described tomodulate tumor microenvironment, leading to a growth advantage forcancer (Savaskan and Eyupoglu, 2010). SLC7A11 was described to beinvolved in neurodegenerative processes in glioma, setting SLC7A11 apotential prime target for cancer therapy (Savaskan et al., 2015).SLC7A11 was shown to be repressed by p53 in the context of ferroptosis,and the p53-SLC7A11 axis was described as preserved in the p53(3KR)mutant, and contributes to its ability to suppress tumorigenesis in theabsence of the classical tumor suppression mechanisms (Jiang et al.,2015). SLC7A11 was described as the functional subunit of systemXc—whose function is increased in aggressive breast cancer cells(Linher-Melville et al., 2015). High membrane staining for SLC7A11 incisplatin-resistant bladder cancer was shown to be associated with apoorer clinical outcome and SLC7A11 inhibition was described as apromising therapeutic approach to the treatment of this disease (Draytonet al., 2014). SLC7A11 was shown to be differentially expressed in thehuman promyelocytic leukemia cell line HL-60 that had been exposed tobenzene and its metabolites and thus highlights a potential associationof SLC7A11 with leukemogenesis (Sarma et al., 2011). Disruption ofSLC7A11 was described to result in growth inhibition of a variety ofcarcinomas, including lymphoma, glioma, prostate and breast cancer (Chenet al., 2009). Inhibition of SLC7A11 was shown to inhibit cell invasionin the esophageal cancer cell line KYSE150 in vitro and its experimentalmetastasis in nude mice and thus establishes a role of SLC7A11 in tumormetastasis (Chen et al., 2009).

SRPR was shown to be amplified in a case of acute myeloid leukemia withdouble minute chromosomes (Crossen et al., 1999).

The human ortholog of SSR4 was shown to be differentially expressed inthe opossum melanoma cell lines TD6b and TD15L2 and up-regulated intumors of advanced stages, implicating SSR4 as a candidate gene withpotential functions that might be associated with ultraviolet-inducedmelanomagenesis and metastasis (Wang and VandeBerg, 2004). The mRNAlevel of SSR4 was shown to be enriched in the osteosarcoma cell linesOHS, SaOS-2 and KPDXM compared to normal osteoblast cells (Olstad etal., 2003).

De-regulated expression of STK17A is associated with different cancertypes. Decreased expression in cervical and colorectal cancer is relatedto the pro-apoptotic character of STK17A connected with tumorprogression. STK17A in glioblastoma and head and neck cancer isover-expressed in a grade-dependent manner, maybe caused through theinfluence on other tumor relevant pathways like TGF-beta (Mao et al.,2013; Thomas et al., 2013; Park et al., 2015; Bandres et al., 2004).STK17A is a direct target of the tumor suppressor gene p53 and amodulator of reactive oxygen species (ROS) (Kerley-Hamilton et al.,2005; Mao et al., 2011).

SYK was described as a modulator of tumorigenesis which acts as a tumorpromoter, by providing a survival function, in some cells and as a tumorsuppressor, by restricting epithelial-mesenchymal transition andinhibiting migration, in others (Krisenko and Geahlen, 2015). SYK wasdescribed as being associated with B-cell receptor (BCR) activation inB-cell lymphomas (Seda and Mraz, 2015). Inhibition of key kinases of theBCR pathway such as SYK have been found in preclinical models todecrease chronic lymphocytic leukemia cell viability (Davids and Brown,2012). SYK was shown to be up-regulated in chronic lymphocytic leukemia(Feng and Wang, 2014). SYK was described as being associated with thepathogenesis of chronic lymphocytic leukemia and might have a value inevaluating the effect of therapy and the prognosis of this disease (Fengand Wang, 2014). SYK was described as a potential tumor suppressor inbreast cancer whose absence in primary breast tumors is correlated withpoor outcomes (Navara, 2004). SYK was shown to play a critical role inpaclitaxel resistance in ovarian cancer (Yu et al., 2015b). SYKdown-regulation was described as being associated with the developmentof various cancers, including colorectal cancer (Peng et al., 2015).Distinct polymorphisms in the SYK promoter were shown to be independentrisk factor for colorectal cancer development in Han Chinese in SouthernChina (Peng et al., 2015). SYK was shown to be frequently methylated inhepatocellular carcinoma and SYK methylation has been demonstrated toidentify a subset of hepatocellular carcinoma cases with poor prognosis(Shin et al., 2014).

TP63 translocation was described as an event in a subset of anaplasticlymphoma kinase-positive anaplastic large cell lymphomas which isassociated with an aggressive course of the disease (Hapgood and Savage,2015). TP63 was described to play a complex role in cancer due to itsinvolvement in epithelial differentiation, cell cycle arrest andapoptosis (Lin et al., 2015). The TP63 isoform TAp63 was described to beover-expressed in hematological malignancies while TP63 missensemutations have been reported in squamous cancers and TP63 translocationsin lymphomas and some lung adenocarcinomas (Orzol et al., 2015).Aberrant splicing resulting in the over-expression of the TP63 isoformDeltaNp63 was described to be frequently found in human cancers such ascutaneous squamous cell carcinoma, where it is likely to favor tumorinitiation and progression (Missero and Antonini, 2014; Inoue and Fry,2014).

TPM1 was shown to be down-regulated in renal cell carcinoma, a squamouscell carcinoma of esophagus cell line, metastatic canine mammarycarcinomas and neuroblastoma cell lines (Klopfleisch et al., 2010; Yageret al., 2003; Zare et al., 2012; Wang et al., 2015b). TPM1 expressionwas shown to be associated with tumor size, Fuhrman grade and theprognosis of renal cell carcinoma patients. TPM1 transfection of renalcell carcinoma cell lines OSRC-2 and 786-0 was shown to reduce migratoryand invasive abilities, while enhancing apoptosis (Wang et al., 2015b).Thus, TPM1 was described to exhibit characteristics of a tumorsuppressor gene while being over-expressed in renal cell carcinoma cells(Wang et al., 2015b). RAS/PI3K/AKT and RAS/MEK/ERK signaling pathwayswere described to be involved in TPM1 regulation and suppression in theintrahepatic cholangiocarcinoma cell line HuCCT1, as well as in a cellline of squamous carcinoma of the esophagus (Zare et al., 2012; Yang etal., 2013b). TPM1 was described as a tumor suppressor whoseover-expression in the breast cancer cell line MCF-7 suppressedanchorage-independent cell growth (Zhu et al., 2007b). Epigeneticsuppression of TPM1 was described to be associated with altered TGF-betatumor suppressor function and might contribute to metastatic propertiesof tumor cells (Varga et al., 2005).

Tryptase was shown to be up-regulated in certain patients with acutemyeloid leukemia (Jin et al., 2014). Expression of tryptase wasdescribed to be regulated by SCF/C-KIT signaling via the ERK1/2 andp38MAPK pathways (Jin et al., 2014). Mast cell tryptase was described tobe involved in colorectal cancer angiogenesis and was shown to be higherexpressed in the serum of colorectal cancer patients before than afterradical surgical resection (Ammendola et al., 2014).

TSHZ3 was shown to be down-regulated in the oral squamous cell carcinomacell line SCC-9 compared to the non-tumorigenic cell line OKF6-TERT1R(Marcinkiewicz and Gudas, 2014). TSHZ3 was described as atranscriptional regulator gene which was found to be recurrentlyrearranged in several cases of high-grade serous ovarian cancers(McBride et al., 2012). TSHZ3 was described as a candidate tumorsuppressor gene with down-regulated expression in breast and prostatecancers (Yamamoto et al., 2011). TSPAN10 was shown to be adifferentially expressed gene between metastatic melanoma samples andnormal skin samples which may be a potential biomarker for metastaticmelanoma therapy (Liu et al., 2014c). Among other genes, TSPAN10 wasshown to be up-regulated in uterine leiomyosarcoma metastases comparedto primary leiomyosarcomas and is therefore contributing to thedifferentiation of these conditions and may aid in understanding tumorprogression in this cancer (Davidson et al., 2014).

TTPAL was described as a candidate oncogene which displayed mutations inmicro-satellite-instable colorectal cancers (Tuupanen et al., 2014).

TUBGCP2 was shown to be up-regulated in a taxol-resistant ovarian cancercell line and was described to be associated with the sensitization ofthe non-small cell lung carcinoma cell line NCI-H1155 to taxol (Huangand Chao, 2015). TUBGCP2 was shown to be up-regulated in glioblastoma,where its over-expression antagonized the inhibitory effect of the CDK5regulatory subunit-associated tumor suppressor protein 3 on DNA damageG2/M checkpoint activity (Draberova et al., 2015).

VIM was described as a down-stream target of STAT3 which is associatedwith breast tumor progression upon de-regulation through STAT3 (Banerjeeand Resat, 2015). VIM was described as a potential nasopharyngealcarcinoma-related protein (Chen et al., 2015c). A negative methylationstatus of vimentin was shown to predict improved prognosis in pancreaticcancer patients (Zhou et al., 2014). VIM was shown to be up-regulatedthrough C6orf106 in non-small cell lung cancer and was described to besubsequently associated with enhanced cancer cell invasion (Zhang etal., 2015c). VIM was described as an independent predictor for overallsurvival of squamous cell lung carcinoma patients (Che et al., 2015).VIM was described as a biomarker that can potentially distinguishmelanoma subtypes and might predict melanoma aggressiveness in differentsubgroups of melanoma (Qendro et al., 2014). VIM was shown to beup-regulated in clear cell renal cell carcinoma (Shi et al., 2015). Highexpression of VIM was described as an independent prognostic indicatorfor clear cell renal cell carcinoma (Shi et al., 2015). VIM was shown toact as a scaffold to recruit Slug to ERK and to promote Slugphosphorylation, which was described as a requirement of the initiationof the epithelial-mesenchymal transition, a developmental processadopted during tumorigenesis that promotes metastatic capacity(Virtakoivu et al., 2015).

WDR1 was shown to be up-regulated in the interstitial fluid from ovariancarcinomas and in high-grade canine cutaneous mast cell tumors with poorprognosis compared to low-grade mast cell tumors with good prognosis(Schlieben et al., 2012; Haslene-Hox et al., 2013). WDR1 was shown to bedown-regulated in chemoresistant advanced serous epithelial ovariancarcinoma (Kim et al., 2011). WDR1 down-regulation in chemoresistantadvanced serous epithelial ovarian carcinoma was shown to be correlatedwith poor overall survival (Kim et al., 2011). WDR1 was shown to beup-regulated in the region between the invading tumor front and normaltissues (interface zone) in breast cancer and thus may be related toprogression and metastasis of breast carcinomas (Kang et al., 2010).

YWHAE fusion with NUTM2B/NUTM2E was described as an event which wasobserved in a minority of clear cell sarcomas of the kidney (Karlsson etal., 2015). The YWHAE-NUTM2 fusion was described as a frequent event inhigh-grade endometrial stromal sarcomas (Ali et al., 2014). High-gradeendometrial stromal sarcomas with the YWHAE-NUTM2 fusion were describedas a subset of endometrial stromal sarcomas with an aggressive clinicalbehavior and poor prognosis (Kruse et al., 2014). Breakages at threeloci including YWHAE were described as potential contributors to thedevelopment of uterine angiosarcoma (Suzuki et al., 2014). YWHAE wasshown to be down-regulated in gastric cancer and reduced YWHAE levelswere associated with diffuse-type gastric cancer and early-onset of thispathology, indicating that YWHAE may have a role in the gastriccarcinogenesis process (Leal et al., 2012). YWHAE was shown to bedifferentially expressed in tissues of breast cancer patients with andwithout relapse and was shown to be associated with both disease-freeand overall survival (Cimino et al., 2008). Thus, YWHAE might be used asan independent prognostic marker and a potential drug target for breastcancer (Cimino et al., 2008).

Changes in ZNF292 were described as chronic lymphocytic leukemia driveralterations (Puente et al., 2015). ZNF292 was described as atumor-suppressor gene in colorectal cancer (Takeda et al., 2015). ZNF292was described as an immunogenic antigen with clinical relevance in headand neck squamous cell carcinoma (Heubeck et al., 2013).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13, or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

Table 5: Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F=1−(1−Gf)².Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004).

Calculated phenotype from allele Allele Population frequency A*02Caucasian (North America)  49.1% A*02 African American (North America) 34.1% A*02 Asian American (North America)  43.2% A*02 Latin American(North American)  48.3% DR1 Caucasian (North America)  19.4% DR2Caucasian (North America)  28.2% DR3 Caucasian (North America)  20.6%DR4 Caucasian (North America)  30.7% DR5 Caucasian (North America) 23.3% DR6 Caucasian (North America)  26.7% DR7 Caucasian (NorthAmerica)  24.8% DR8 Caucasian (North America)  5.7% DR9 Caucasian (NorthAmerica)  2.1% DR1 African (North) American 13.20% DR2 African (North)American 29.80% DR3 African (North) American 24.80% DR4 African (North)American 11.10% DR5 African (North) American 31.10% DR6 African (North)American 33.70% DR7 African (North) American 19.20% DR8 African (North)American 12.10% DR9 African (North) American  5.80% DR1 Asian (North)American  6.80% DR2 Asian (North) American 33.80% DR3 Asian (North)American  9.20% DR4 Asian (North) American 28.60% DRS Asian (North)American 30.00% DR6 Asian (North) American 25.10% DR7 Asian (North)American 13.40% DR8 Asian (North) American 12.70% DR9 Asian (North)American 18.60% DR1 Latin (North) American 15.30% DR2 Latin (North)American 21.20% DR3 Latin (North) American 15.20% DR4 Latin (North)American 36.80% DRS Latin (North) American 20.00% DR6 Latin (North)American 31.10% DR7 Latin (North) American 20.20% DR8 Latin (North)American 18.60% DR9 Latin (North) American  2.10% A*24 Philippines   65%A*24 Russia Nenets   61% A*24:02 Japan   59% A*24 Malaysia   58% A*24:02Philippines   54% A*24 India   47% A*24 South Korea   40% A*24 Sri Lanka  37% A*24 China   32% A*24:02 India   29% A*24 Australia West   22%A*24 USA   22% A*24 Russia Samara   20% A*24 South America   20% A*24Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences; and R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity, then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 93, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 93. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells. These T cells can subsequently cross-react with cellsand kill cells that express a polypeptide that contains the naturalamino acid sequence of the cognate peptide as defined in the aspects ofthe invention. As can be derived from the scientific literature anddatabases (Rammensee et al., 1999; Godkin et al., 1997), certainpositions of HLA binding peptides are typically anchor residues forminga core sequence fitting to the binding motif of the HLA receptor, whichis defined by polar, electrophysical, hydrophobic and spatial propertiesof the polypeptide chains constituting the binding groove. Thus, oneskilled in the art would be able to modify the amino acid sequences setforth in SEQ ID NO: 1 to SEQ ID NO: 93, by maintaining the known anchorresidues, and would be able to determine whether such variants maintainthe ability to bind MHC class I or II molecules. The variants of thepresent invention retain the ability to bind to the TCR of activated Tcells, which can subsequently cross-react with and kill cells thatexpress a polypeptide containing the natural amino acid sequence of thecognate peptide as defined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gin); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 5—large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according to SEQ ID NO: 4, 9,and 18 Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No.: 4 A L F G T I L E LVariants V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A QV Q I Q Q A SEQ ID No.: 9 H L I A E I H T A Variants V I L M V M I M L MA V A I A L A V V V I V L V T V T I T L T Q V Q I Q L Q SEQ ID No.: 18 FL L D Q V Q L G L Variants V I A M V M I M M A A V A I A A A V V V I V VA T V T I T T A Q V Q I Q Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 93.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 93 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from esophageal cancersamples (N=16 A*02-positive samples) with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from 16 esophageal cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from esophageal cancer tissue samples werepurified and HLA-associated peptides were isolated and analyzed by LC-MS(see examples). All TUMAPs contained in the present application wereidentified with this approach on primary esophageal cancer samplesconfirming their presentation on primary esophageal cancer.

TUMAPs identified on multiple esophageal cancer and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

In addition to over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (see Example 2). An additionalsource of normal tissue data was a database of publicly available RNAexpression data from around 3000 normal tissue samples (Lonsdale, 2013).Peptides which are derived from proteins whose coding mRNA is highlyexpressed in cancer tissue, but very low or absent in vital normaltissues, were preferably included in the present invention.

Furthermore, the discovery pipeline XPRESIDENT® v2. allows for a directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 12).

The present invention provides peptides that are useful in treatingcancers/tumors, preferably esophageal cancer that over- or exclusivelypresent the peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanesophageal cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy esophagus cells or other normal tissue cells, demonstrating ahigh degree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from esophageal cancer, but not on normaltissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. esophageal cancer cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are HAVCR1-001 peptides capable of binding to TCRs andantibodies when presented by an MHC molecule. The present descriptionalso relates to nucleic acids, vectors and host cells for expressingTCRs and peptides of the present description; and methods of using thesame.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an HAVCR1-001peptide-HLA molecule complex with a binding affinity (KD) of about 100μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM orless. More preferred are high affinity TCRs having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less. Non-limiting examples of preferred binding affinity rangesfor TCRs of the present invention include about 1 nM to about 10 nM;about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM toabout 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM;about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM toabout 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for an HAVCR1-001 peptide-HLAmolecule complex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the non-mutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a HAVCR1-001 peptide-HLA molecule complex, whichis at least double that of a TCR comprising the non-mutated TCR alphachain and/or non-mutated TCR beta chain. Affinity-enhancement oftumor-specific TCRs, and its exploitation, relies on the existence of awindow for optimal TCR affinities. The existence of such a window isbased on observations that TCRs specific for HLA-A2-restricted pathogenshave KD values that are generally about 10-fold lower when compared toTCRs specific for HLA-A2-restricted tumor-associated self-antigens. Itis now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to HAVCR1-001 can be enhanced by methods well known in theart.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/HAVCR1-001 monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRap geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with HAVCR1-001, incubating PBMCs obtained from the transgenicmice with tetramer-phycoerythrin (PE), and isolating the high avidityT-cells by fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems.

The in vitro-synthesized TCR RNAs are then introduced into primary CD8+T-cells obtained from healthy donors by electroporation to re-expresstumor specific TCR-alpha and/or TCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intra-ribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3 (CD3 fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 93, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the fl origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbas and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used.

With regard to transformation of prokaryotic host cells, see, forexample, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook,2012). Transformation of yeast cells is described in Sherman et al.(Sherman et al., 1986). The method of Beggs (Beggs, 1978) is alsouseful. With regard to vertebrate cells, reagents useful in transfectingsuch cells, for example calcium phosphate and DEAE-dextran or liposomeformulations, are available from Stratagene Cloning Systems, or LifeTechnologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is alsouseful for transforming and/or transfecting cells and is well known inthe art for transforming yeast cell, bacterial cells, insect cells andvertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid coglycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation. Preferred adjuvants are anti-CD40, imiquimod,resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab,interferon-alpha, CpG oligonucleotides and derivates, poly-(I:C) andderivates, RNA, sildenafil, and particulate formulations with PLG orvirosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one Ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO: 93,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 93, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 93 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:93 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 93, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 93.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells. Another embodiment of the presentinvention relates to a non-naturally occurring peptide wherein saidpeptide consists or consists essentially of an amino acid sequenceaccording to SEQ ID No: 1 to SEQ ID No: 48 and has been syntheticallyproduced (e.g. synthesized) as a pharmaceutically acceptable salt.Methods to synthetically produce peptides are well known in the art. Thesalts of the peptides according to the present invention differsubstantially from the peptides in their state(s) in vivo, as thepeptides as generated in vivo are no salts. The non-natural salt form ofthe peptide mediates the solubility of the peptide, in particular in thecontext of pharmaceutical compositions comprising the peptides, e.g. thepeptide vaccines as disclosed herein. A sufficient and at leastsubstantial solubility of the peptide(s) is required in order toefficiently provide the peptides to the subject to be treated.Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn₂ ⁺, Mg₂ ⁺, Ca₂ ⁺, Mn₂ ⁺,Cu₂ ⁺ and Ba₂ ⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(N₀₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrine, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of esophageal cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 93 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are esophageal cancer cells orother solid or hematological tumor cells such as lung cancer, urinarybladder cancer, ovarian cancer, melanoma, uterine cancer, hepatocellularcancer, renal cell cancer, brain cancer, colorectal cancer, breastcancer, gastric cancer, pancreatic cancer, gallbladder cancer, bile ductcancer, prostate cancer and leukemia.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of esophageal cancer. The present invention also relates tothe use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a esophageal cancer marker(poly)peptide, delivery of a toxin to a esophageal cancer cellexpressing a cancer marker gene at an increased level, and/or inhibitingthe activity of a esophageal cancer marker polypeptide) according to theinvention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length esophageal cancer marker polypeptides orfragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 93polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the esophageal cancer markerpolypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating esophagealcancer, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occur in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.

Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 93, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition, plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 93.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal (healthy)tissues or that the gene is silent in the tissue from which the tumor isderived but in the tumor it is expressed. By “over-expressed” theinventors mean that the polypeptide is present at a level at least1.2-fold of that present in normal tissue; preferably at least 2-fold,and more preferably at least 5-fold or 10-fold the level present innormal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from esophagealcancer, the medicament of the invention is preferably used to treatesophageal cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofesophageal cancer patients with various HLA-A HLA-B and HLA-C alleles.It may contain MHC class I and MHC class II peptides or elongated MHCclass I peptides. In addition to the tumor associated peptides collectedfrom several esophageal cancer tissues, the warehouse may containHLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparisonof the magnitude of T-cell immunity induced by TUMAPS in a quantitativemanner and hence allow important conclusion to be drawn on the capacityof the vaccine to elicit anti-tumor responses. Secondly, they functionas important positive control peptides derived from a “non-self” antigenin the case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, esophageal cancer samples frompatients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue(esophageal cancer) compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromesophageal cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of 2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from esophageal cancer cells and since it was determinedthat these peptides are not or at lower levels present in normaltissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for esophageal cancer. Presence of groups of peptides canenable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A to 1V show the over-presentation of various peptides in normaltissues (white bars) and esophageal cancer (black bars). FIG. 1A) Genesymbol: KRT14/KRT16, Peptide: STYGGGLSV (SEQ ID NO: 1) Tissues from leftto right: 1 adipose tissues, 3 adrenal glands, 8 arteries, 5 bonemarrows, 7 brains, 5 breasts, 2 cartilages, 1 central nerve, 13 colons,1 duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4lymph nodes, 4 leukocyte samples, 3 ovaries, 8 pancreas, 5 peripheralnerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3prostates, 6 recti, 7 salivary glands, 4 skeletal muscles, 6 skins, 2small intestines, 4 spleens, 5 stomachs, 6 testis, 3 thymi, 3 thyroidglands, 7 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 2 veins, 6esophagi, 16 esophageal cancer samples. The peptide has additionallybeen detected on 4/91 lung cancers. FIG. 1B) Gene symbol: GJB5, Peptide:SIFEGLLSGV (SEQ ID NO: 7). Tissues from left to right: 1 adiposetissues, 3 adrenal glands, 8 arteries, 5 bone marrows, 7 brains, 5breasts, 2 cartilages, 1 central nerve, 13 colons, 1 duodenum, 2gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes,4 leukocyte samples, 3 ovaries, 8 pancreas, 5 peripheral nerves, 1peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3 prostates, 6recti, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 smallintestines, 4 spleens, 5 stomachs, 6 testis, 3 thymi, 3 thyroid glands,7 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 2 veins, 6 esophagi,16 esophageal cancer samples. The peptide has additionally been detectedon 1/43 prostate cancers, 1/3 gallbladder cancers, 1/20 ovarian cancers,5/91 lung cancers and 1/4 urinary bladder cancers. FIG. 1C) Gene symbol:PKP3, Peptide: SLVSEQLEPA (SEQ ID NO: 34). Tissues from left to right: 1adipose tissues, 3 adrenal glands, 8 arteries, 5 bone marrows, 7 brains,5 breasts, 2 cartilages, 1 central nerve, 13 colons, 1 duodenum, 2gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4 lymph nodes,4 leukocyte samples, 3 ovaries, 8 pancreas, 5 peripheral nerves, 1peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3 prostates, 6recti, 7 salivary glands, 4 skeletal muscles, 6 skins, 2 smallintestines, 4 spleens, 5 stomachs, 6 testis, 3 thymi, 3 thyroid glands,7 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 2 veins, 6 esophagi,16 esophageal cancer samples. The peptide has additionally been detectedon 8/24 colorectal cancers, 1/20 ovarian cancers, 1/46 gastric cancers,5/91 lung cancers and 2/4 urinary bladder cancers. FIG. 1D) Gene symbol:RNPEP, Peptide: YTQPFSHYGQAL (SEQ ID NO: 37). Tissues from left toright: 1 adipose tissues, 3 adrenal glands, 8 arteries, 5 bone marrows,7 brains, 5 breasts, 2 cartilages, 1 central nerve, 13 colons, 1duodenum, 2 gallbladders, 5 hearts, 14 kidneys, 21 livers, 44 lungs, 4lymph nodes, 4 leukocyte samples, 3 ovaries, 8 pancreas, 5 peripheralnerves, 1 peritoneum, 3 pituitary glands, 4 placentas, 3 pleuras, 3prostates, 6 recti, 7 salivary glands, 4 skeletal muscles, 6 skins, 2small intestines, 4 spleens, 5 stomachs, 6 testis, 3 thymi, 3 thyroidglands, 7 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 2 veins, 6esophagi, 16 esophageal cancer samples. The peptide has additionallybeen detected on 1/19 pancreatic cancers, 7/46 gastric cancers and 1/91lung cancers. FIG. 1E) Gene symbol: NUP155, Peptide: ALQEALENA (SEQ IDNO: 80). Samples from left to right: 4 cell lines (1 kidney, 1pancreatic, 1 prostate, 1 myeloid leukemia), 3 normal tissues (1 lung, 1prostate, 1 small intestine), 47 cancer tissues (5 brain cancers, 2breast cancers, 1 colon cancers, 2 esophageal cancers, 1 chronicleukocytic leukemia, 2 liver cancers, 22 lung cancers, 7 ovariancancers, 4 prostate cancers, 1 rectum cancer). FIG. 1F) Gene symbol:KRT5, Peptide: SLYNLGGSKRISI (SEQ ID NO: 2). Tissues from left to right:20 cancer tissues (9 head-and-neck cancers, 2 esophageal cancers, 1esophagus and stomach cancer, 7 lung cancers, 1 urinary bladder cancer).FIG. 1G) Gene symbol: KRT5, Peptide: TASAITPSV (SEQ ID NO: 3). Tissuesfrom left to right: 17 cancer tissues (2 esophageal cancers, 6head-and-neck cancers, 7 lung cancers, 2 urinary bladder cancers). FIG.1H) Gene symbol: S100A2, Peptide: SLDENSDQQV (SEQ ID NO: 10). Tissuesfrom left to right: 7 cancer tissues (3 head-and-neck cancers, 2esophageal cancers, 1 lung cancer, 1 urinary bladder cancer). FIG. 1I)Gene symbol: LAMB3, Peptide: ALWLPTDSATV (SEQ ID NO: 11). Tissues fromleft to right: 12 cancer tissues (2 esophageal cancers, 1 gallbladdercancer, 8 lung cancers, 1 skin cancer). FIG. 1J) Gene symbol: IL36RN,Peptide: SLSPVILGV (SEQ ID NO: 13). Tissues from left to right: 26cancer tissues (8 head-and-neck cancers, 3 esophageal cancers, 10 lungcancers, 3 skin cancers, 1 urinary bladder cancer, 1 uterus cancer).FIG. 1K) Gene symbol: ANO1, Peptide: LLANGVYAA (SEQ ID NO: 15). Tissuesfrom left to right: 8 cancer tissues (2 esophageal cancers, 1gallbladder cancer, 1 liver cancer, 1 lung cancer, 1 stomach cancer, 1urinary bladder cancer, 1 uterus cancer). FIG. 1L) Gene symbol: F7,IGHV4-31, IGHG1, IGHG2, IGHG3, IGHG4, IGHM, Peptide: MISRTPEV (SEQ IDNO: 17). Tissues from left to right: 19 cancer tissues (2 esophagealcancers, 2 kidney cancers, 2 liver cancers, 9 lung cancers, 1 lymph nodecancer, 1 testis cancer, 2 urinary bladder cancers. FIG. 1M) Genesymbol: QSER1, Peptide: SLNGNQVTV (SEQ ID NO: 30). Tissues from left toright: 1 cell line (1 pancreatic), 14 cancer tissues (1 head-and-neckcancer, 1 bile duct cancer, 1 brain cancer, 1 breast cancer, 1esophageal cancer, 1 kidney cancer, 1 lung cancer, 2 skin cancers, 3urinary bladder cancers, 2 uterus cancers). FIG. 1N) Gene symbol: HAS3,Peptide: YMLDIFHEV (SEQ ID NO: 32). Tissues from left to right: 1 normaltissue (1 uterus), 15 cancer tissues (1 brain cancer, 2 esophagealcancers, 1 gallbladder cancer, 3 head-and-neck cancers, 4 lung cancers,4 urinary bladder cancers). FIG. 1O) Gene symbol: PKP3, Peptide:SLVSEQLEPA (SEQ ID NO: 34). Tissues from left to right: 1 cell line (1pancreatic), 1 normal tissue (1 colon), 28 cancer tissues (6head-and-neck cancers, 1 breast cancer, 1 cecum cancer, 3 colon cancers,1 colorectal cancer, 3 esophageal cancers, 6 lung cancers, 1 ovariancancer, 3 rectum cancers, 3 urinary bladder cancers). FIG. 1P) Genesymbol: SERPINH1, Peptide: GLAFSLYQA (SEQ ID NO: 40). Tissues from leftto right: 3 cell lines (1 kidney, 2 pancreatic), 4 normal tissues (1adrenal gland, 1 lung, 2 placentas), 41 cancer tissues (3 head-and-neckcancers, 3 breast cancers, 2 colon cancers, 2 esophageal cancers, 1gallbladder cancer, 1 liver cancer, 15 lung cancers, 1 ovarian cancer, 1pancreas cancer, 3 rectum cancers, 2 skin cancers, 1 stomach cancer, 4urinary bladder cancers, 2 uterus cancers). FIG. 1Q) Gene symbol:TMEM132A, Peptide: ALVEVTEHV (SEQ ID NO: 56). Tissues from left toright: 7 normal tissues (5 lungs, 1 thyroid gland, 1 trachea), 64 cancertissues (6 head-and-neck cancers, 12 brain cancers, 4 breast cancers, 3esophageal cancers, 1 gallbladder cancer, 5 kidney cancers, 21 lungcancers, 1 lymph node cancer, 7 ovarian cancers, 1 pancreas cancer, 1skin cancer, 2 uterus cancers). FIG. 1R) Gene symbol: PRC1, Peptide:GLAPNTPGKA (SEQ ID NO: 57). Tissues from left to right: 14 cancertissues (1 head-and-neck cancer, 1 breast cancer, 2 esophageal cancers,6 lung cancers, 1 ovarian cancer, 1 skin cancer, 1 urinary bladdercancer, 1 uterus cancer). FIG. 1S) Gene symbol: MAPK6, Peptide:LILESIPVV (SEQ ID NO: 58). Tissues from left to right: 2 cell lines (1blood cell, 1 skin), 25 cancer tissues (5 head-and-neck cancers, 1 coloncancer, 2 esophageal cancers, 1 leukocytic leukemia cancer, 8 lungcancers, 2 lymph node cancers, 3 skin cancers, 2 urinary bladdercancers, 1 uterus cancer). FIG. 1T) Gene symbol: PPP4R1, Peptide:SLLDTLREV (SEQ ID NO: 59). Tissues from left to right: 1 normal tissue(1 small intestine), 8 cancer tissues (1 head-and-neck cancer, 2esophageal cancers, 4 lung cancers, 1 ovarian cancer). FIG. 1U) Genesymbol: TP63, Peptide: VLVPYEPPQV (SEQ ID NO: 77). Tissues from left toright: 2 normal tissues (1 esophagus, 1 trachea), 47 cancer tissues (8head-and-neck cancers, 4 esophageal cancers, 1 gallbladder cancer, 14lung cancers, 7 lymph node cancers, 2 prostate cancers, 1 skin cancer, 8urinary bladder cancers. FIG. 1V) Gene symbol: KIAA0947, Peptide:AVLPHVDQV (SEQ ID NO: 81). Tissues from left to right: 3 cell lines (1blood cells, 1 pancreatic), 12 cancer tissues (5 brain cancers, 2esophageal cancers, 1 lung cancer, 3 lymph node cancers, 1 uteruscancer).

FIGS. 2A to 2D show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in esophageal cancer in a panel of normal tissues (white bars)and 11 esophageal cancer samples (black bars). Tissues from left toright: 7 arteries, 1 brain, 1 heart, 2 livers, 2 lungs, 2 veins, 1adipose tissue, 1 adrenal gland, 4 bone marrows, 1 colon, 2 esophagi, 2gallbladders, 1 kidney, 6 lymph nodes, 1 pancreas, 1 pituitary gland, 1rectum, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thymus, 1 thyroid gland, 5 tracheae, 1 urinary bladder, 1breast, 3 ovaries, 3 placentae, 1 prostate, 1 testis, 1 uterus, 11esophageal cancer samples. FIG. 2A) Gene symbol: PTHLH; FIG. 2B) Genesymbol: KRT14; FIG. 2C) Gene symbol: FAM83A; FIG. 2D) Gene symbol: PDPN.

FIGS. 3A to 3E show exemplary results of peptide-specific in vitro CD8+T cell responses of a healthy HLA-A*02+ donor i.e. exemplaryimmunogenicity data: flow cytometry results after peptide-specificmultimer staining. FIG. 3A) Gene symbol: SF3B3, Peptide: ELDRTPPEV (SEQID NO: 97); FIG. 3B) Gene symbol: TNC, Peptide: AMTQLLAGV (SEQ ID NO:101). Also, CD8+ T cells were primed using artificial APCs coated withanti-CD28 mAb and HLA-A*02 in complex with SEQ ID NO: 5 peptide (FIG.3C, left panel), SEQ ID NO: 2 peptide (FIG. 3D, left panel) and SEQ IDNO: 77 peptide (FIG. 3E, left panel), respectively. After three cyclesof stimulation, the detection of peptide-reactive cells was performed by2D multimer staining with A*02/Seq ID No 5 (FIG. 3C), A*02/Seq ID No 2(FIG. 3D) or A*02/Seq ID No 77 (FIG. 3E). Right panels (FIGS. 3C, 3D and3E) show control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from Asterand (Detroit, USA andRoyston, Herts, UK); ProteoGenex Inc., (Culver City, Calif., USA);Tissue Solutions Ltd. (Glasgow, UK); University Hospital of Tubingen.Normal tissues were obtained from Asterand (Detroit, USA and Royston,Herts, UK); Bio-Options Inc. (CA, USA); BioServe (Beltsville, Md., USA);Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); University Hospital of Geneva; UniversityHospital of Heidelberg; Kyoto Prefectural University of Medicine (KPUM);University Hospital Munich; ProteoGenex Inc. (Culver City, Calif., USA);University Hospital of Tubingen; Tissue Solutions Ltd. (Glasgow, UK).Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose esophageal cancer samples to a baseline of normaltissue samples.

Presentation profiles of exemplary over-presented peptides are shown inFIGS. 1A-1V. Presentation scores for exemplary peptides are shown inTable 8.

Table 8: Presentation scores. The table lists peptides that are veryhighly over-presented on tumors compared to a panel of normal tissues(+++), highly over-presented on tumors compared to a panel of normaltissues (++) or over-presented on tumors compared to a panel of normaltissues (+). The panel of normal tissues consisted of: adipose tissue,adrenal gland, artery, vein, bone marrow, brain, central and peripheralnerve, colon, rectum, small intestine incl. duodenum, esophagus,gallbladder, heart, kidney, liver, lung, lymph node, mononuclear whiteblood cells, pancreas, peritoneum, pituitary, pleura, salivary gland,skeletal muscle, skin, spleen, stomach, thymus, thyroid gland, trachea,ureter, urinary bladder.

SEQ ID No. Sequence Peptide Presentation 1 STYGGGLSV +++ 2 SLYNLGGSKRISI+++ 3 TASAITPSV +++ 4 ALFGTILEL ++ 5 NLMASQPQL +++ 6 LLSGDLIFL +++ 7SIFEGLLSGV +++ 8 ALLDGGSEAYWRV +++ 9 HLIAEIHTA +++ 10 SLDENSDQQV +++ 11ALWLPTDSATV +++ 12 GLASRILDA +++ 13 SLSPVILGV +++ 14 RLPNAGTQV +++ 15LLANGVYAA +++ 16 VLAEGGEGV +++ 17 MISRTPEV +++ 18 FLLDQVQLGL +++ 19GLAPFLLNAV +++ 20 IIEVDPDTKEML +++ 21 IVREFLTAL +++ 22 KLNDTYVNV +++ 23KLSDSATYL +++ 24 LLFAGTMTV +++ 25 LLPPPPPPA +++ 26 MLAEKLLQA +++ 27NLREGDQLL +++ 28 SLDGFTIQV +++ 29 SLDGTELQL +++ 30 SLNGNQVTV +++ 32YMLDIFHEV +++ 33 GLDVTSLRPFDL +++ 34 SLVSEQLEPA + 35 LLRFSQDNA +++ 36FLLRFSQDNA +++ 37 YTQPFSHYGQAL +++ 38 IAAIRGFLV +++ 39 LVRDTQSGSL +++ 40GLAFSLYQA +++ 41 GLESEELEPEEL + 44 ATGNDRKEAAENSL +++ 45 MLTELEKAL +++47 VLASGFLTV +++ 48 SMHQMLDQTL +++ 50 GMNPHQTPAQL +++ 51 KLFGHLTSA +++52 VAIGGVDGNVRL +++ 55 GAIDLLHNV +++ 57 GLAPNTPGKA +++ 58 LILESIPVV +++59 SLLDTLREV +++ 61 TQTTHELTI +++ 62 ALYEYQPLQI +++ 63 LAYTLGVKQL +++ 64GLTDVIRDV ++ 65 YVVGGFLYQRL +++ 66 LLDEKVQSV + 68 PAVLQSSGLYSL +++ 70FVLDTSESV + 71 ASDPILYRPVAV + 72 FLPPAQVTV + 73 KITEAIQYV + 75GLMDDVDFKA + 77 VLVPYEPPQV ++ 78 KVANIIAEV + 80 ALQEALENA ++ 81AVLPHVDQV +++ 82 HLLGHLEQA +++ 84 SLAESLDQA + 86 GLLTEIRAV + 87FLDNGPKTI + 88 GLWEQENHL + 89 SLADSLYNL + 91 KLIDDVHRL + 92 SILRHVAEV +94 TLLQEQGTKTV +

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from tumor tissue for RNASeq experiments was obtained from:ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd.(Glasgow, UK).

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, USA and Royston, Herts, UK); ProteoGenex Inc.(Culver City, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);Istituto Nazionale Tumori “Pascale”, Molecular Biology and ViralOncology Unit (IRCCS) (Naples, Italy); University Hospital of Heidelberg(Germany); BioCat GmbH (Heidelberg, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (Illumina Inc,San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolarly and sequenced on the IlluminaHiSeq 2500 sequencer according to the manufacturer's instructions,generating 50 bp single end reads. Processed reads are mapped to thehuman genome (GRCh38) using the STAR software. Expression data areprovided on transcript level as RPKM (Reads Per Kilobase per Millionmapped reads, generated by the software Cufflinks) and on exon level(total reads, generated by the software Bedtools), based on annotationsof the ensembl sequence database (Ensembl77). Exon reads are normalizedfor exon length and alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in esophagealcancer are shown in FIGS. 2A-2D. Expression scores for further exemplarygenes are shown in Table 9.

Table 9: Expression scores. The table lists peptides from genes that arevery highly over-expressed in tumors compared to a panel of normaltissues (+++), highly over-expressed in tumors compared to a panel ofnormal tissues (++) or over-expressed in tumors compared to a panel ofnormal tissues (+). The baseline for this score was calculated frommeasurements of the following normal tissues: adipose tissue, adrenalgland, artery, bone marrow, brain, colon, esophagus, gallbladder, heart,kidney, liver, lung, lymph node, pancreas, pituitary, rectum, skeletalmuscle, skin, small intestine, spleen, stomach, thymus, thyroid gland,trachea, urinary bladder, vein.

SEQ ID No. Sequence Gene Expression 1 STYGGGLSV +++ 2 SLYNLGGSKRISI +++3 TASAITPSV +++ 4 ALFGTILEL ++ 5 NLMASQPQL +++ 6 LLSGDLIFL +++ 7SIFEGLLSGV +++ 8 ALLDGGSEAYWRV +++ 9 HLIAEIHTA +++ 10 SLDENSDQQV +++ 11ALWLPTDSATV +++ 12 GLASRILDA +++ 13 SLSPVILGV +++ 14 RLPNAGTQV +++ 15LLANGVYAA +++ 16 VLAEGGEGV +++ 17 MISRTPEV +++ 18 FLLDQVQLGL +++ 24LLFAGTMTV +++ 25 LLPPPPPPA + 26 MLAEKLLQA ++ 27 NLREGDQLL +++ 32YMLDIFHEV +++ 49 GLMKDIVGA + 55 GAIDLLHNV ++ 57 GLAPNTPGKA + 67SMNGGVFAV ++ 69 GLLVGSEKVTM +++ 71 ASDPILYRPVAV + 77 VLVPYEPPQV +++ 80ALQEALENA + 94 TLLQEQGTKTV +++

Example 3 In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nirnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 102) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.103), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Esophageal Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for twopeptides (SEQ ID No 97 and SEQ ID No 101) of the invention are shown inFIGS. 3A-3E together with corresponding negative controls. Results forfive peptides from the invention are summarized in Table 10A.

TABLE 10A  in vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immunogenicityexperiments conducted by theapplicant for the peptides of the invention.  SEQ ID No Sequence wellsDonors 94 TLLQEQGTKTV + ++ 95 LIQDRVAEV + ++ 97 ELDRTPPEV ++ ++++ 98VLFPNLKTV + ++++ 101 AMTQLLAGV ++ +++ <20% = +; 20%-49% = ++; 50%-69% =+++; >=70% = ++++

TABLE 10B  In vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for peptides of the invention.SEQ ID No Sequence Wells positive [%] 1 STYGGGLSV + 2 SLYNLGGSKRISI + 5NLMASQPQL +++ 6 LLSGDLIFL ++ 12 GLASRILDA + 19 GLAPFLLNAV + 29SLDGTELQL + 47 VLASGFLTV +++ 69 GLLVGSEKVTM + 77 VLVPYEPPQV + Results ofin vitro immunogenicity experiments are indicated. Percentage ofpositive wells and donors (among evaluable) are summarized as indicated

Example 4 Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5 MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (p2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11  MHC class I binding scores. SEQ ID Sequence Peptide exchange 1STYGGGLSV +++ 2 SLYNLGGSKRISI ++++ 3 TASAITPSV +++ 5 NLMASQPQL +++ 6LLSGDLIFL +++ 7 SIFEGLLSGV ++ 8 ALLDGGSEAYWRV +++ 9 HLIAEIHTA +++ 10SLDENSDQQV +++ 11 ALWLPTDSATV +++ 12 GLASRILDA +++ 13 SLSPVILGV ++++ 14RLPNAGTQV ++++ 15 LLANGVYAA + 16 VLAEGGEGV +++ 17 MISRTPEV ++ 18FLLDQVQLGL +++ 19 GLAPFLLNAV +++ 21 IVREFLTAL +++ 22 KLNDTYVNV +++ 23KLSDSATYL +++ 24 LLFAGTMTV ++ 25 LLPPPPPPA +++ 26 MLAEKLLQA + 27NLREGDQLL +++ 28 SLDGFTIQV ++ 29 SLDGTELQL +++ 30 SLNGNQVTV + 31VLPKLYVKL ++ 32 YMLDIFHEV ++ 33 GLDVTSLRPFDL +++ 34 SLVSEQLEPA +++ 35LLRFSQDNA +++ 36 FLLRFSQDNA ++ 37 YTQPFSHYGQAL +++ 38 IAAIRGFLV +++ 39LVRDTQSGSL ++ 40 GLAFSLYQA ++ 41 GLESEELEPEEL ++ 42 TQTAVITRI + 43KVVGKDYLL + 44 ATGNDRKEAAENSL +++ 45 MLTELEKAL ++ 46 YTAQIGADIAL +++ 47VLASGFLTV ++++ 48 SMHQMLDQTL ++ 49 GLMKDIVGA +++ 51 KLFGHLTSA ++ 52VAIGGVDGNVRL ++ 53 VVVTGLTLV ++ 54 YQDLLNVKM +++ 55 GAIDLLHNV ++ 56ALVEVTEHV ++ 57 GLAPNTPGKA +++ 58 LILESIPVV ++ 59 SLLDTLREV +++ 60VVMEELLKV ++ 61 TQTTHELTI +++ 62 ALYEYQPLQI ++ 63 LAYTLGVKQL +++ 64GLTDVIRDV ++++ 65 YVVGGFLYQRL +++ 66 LLDEKVQSV +++ 67 SMNGGVFAV ++ 68PAVLQSSGLYSL ++ 69 GLLVGSEKVTM +++ 70 FVLDTSESV +++ 71 ASDPILYRPVAV +++72 FLPPAQVTV ++ 73 KITEAIQYV +++ 74 ILASLATSV +++ 76 KVADYIPQL +++ 77VLVPYEPPQV ++ 78 KVANIIAEV ++ 79 GQDVGRYQV ++ 80 ALQEALENA ++ 81AVLPHVDQV +++ 82 HLLGHLEQA +++ 83 ALADGVVSQA +++ 84 SLAESLDQA +++ 85NIIELVHQV ++++ 87 FLDNGPKTI +++ 89 SLADSLYNL ++ 90 SIYEYYHAL +++ 91KLIDDVHRL ++++ 92 SILRHVAEV ++ 93 VLINTSVTL +++ Binding of HLA-class Irestricted peptides to HLA-A*02:01 was ranged by peptide exchange yield:≥10% = +; ≥20% = ++; ≥50% = +++; ≥75% = ++++

Example 6 Absolute Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and -specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.The quantitation of TUMAP copies per cell in solid tumor samplesrequires the absolute quantitation of the isolated TUMAP, the efficiencyof TUMAP isolation, and the cell count of the tissue sample analyzed.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard; theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell numbers for selected peptides are shownin Table 12.

TABLE 12 Absolute copy numbers. SEQ ID Copies per cell Number of No.Peptide Code (median) samples 9 PTHL-001 + 31 The table lists theresults of absolute peptide quantitation in NSCLC tumor samples. Themedian number of copies per cell are indicated for each peptide: <100 =+; >=100 = ++; >=1,000 +++; >=10,000 = ++++. The number of samples, inwhich evaluable, high quality MS data are available, is indicated.

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1. A method of eliciting an immune response in a patient who has cancer,comprising administering to said patient a population of activated Tcells that selectively recognize cells, which present a peptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 1 to SEQ ID NO: 76 and SEQ ID NO: 78 to SEQ ID NO: 93,wherein the activated T cells are cytotoxic T cells produced bycontacting T cells with an antigen presenting cell that expresses thepeptide in a complex with an MHC molecule on the surface of the antigenpresenting cell, for a period of time sufficient to activate said Tcell, and wherein said cancer is selected from the group consisting oflung cancer, kidney cancer, brain cancer, stomach cancer, colon orrectal cancer, liver cancer, prostate cancer, leukemia, breast cancer,Merkel cell carcinoma (MCC), melanoma, ovarian cancer, esophagealcancer, urinary bladder cancer, endometrial cancer, gall bladder cancer,and bile duct cancer.
 2. The method of claim 1, wherein the T cells areautologous to the patient.
 3. The method of claim 1, wherein the T cellsare obtained from a healthy donor.
 4. The method of claim 1, wherein theT cells are obtained from tumor infiltrating lymphocytes or peripheralblood mononuclear cells.
 5. The method of claim 1, wherein the activatedT cells are expanded in vitro.
 6. The method of claim 1, wherein the MHCmolecule is an MHC class I molecule.
 7. The method of claim 1, whereinthe antigen presenting cell is infected with a recombinant virusexpressing the peptide.
 8. The method of claim 7, wherein the antigenpresenting cell is a dendritic cell or a macrophage.
 9. The method ofclaim 5, wherein the expansion is in the presence of an anti-CD28antibody and IL-12.
 10. The method of claim 1, wherein the population ofactivated T cells comprises CD8-positive cells.
 11. The method of claim1, wherein the population of activated T cells are administered in theform of a composition.
 12. The method of claim 11, wherein thecomposition comprises an adjuvant.
 13. The method of claim 12, whereinthe adjuvant is selected from the group consisting of imiquimod,resiguimod, GM-CSF, cyclophosphamide, Sunitinib, bevacizumab,interferon-alpha, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, and particulate formations withpoly(lactid coglycolid) (PLG) and virosomes.
 14. The method of claim 1,wherein the contacting T cells is in vitro.
 15. The method of claim 1,wherein the immune response is capable of killing the cancer cells. 16.The method of claim 15, wherein the immune response comprises acytotoxic T cell response.
 17. The method of claim 1, wherein the immuneresponse comprises a cytotoxic T cell response.
 18. The method of claim1, wherein the cancer is esophageal cancer.
 19. The method of claim 1,wherein the cancer is non-small cell lung cancer.
 20. The method ofclaim 1, wherein the MHC molecule is HLA-A*02.